Variable regions for NKp46 binding proteins

ABSTRACT

NKp46-binding immunoglobulin variable regions, and proteins such as antibodies and multispecific proteins that comprise the variable regions are provided. The proteins can bind and specifically redirect NK cells to lyse a target cell of interest. The proteins have utility in the treatment of disease, notably cancer or infectious disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/066,688, filed Jun. 28, 2018, now U.S. Pat. No. 11,001,629, which isthe U.S. national stage application of International Patent ApplicationNo. PCT/EP2016/081953, filed Dec. 20, 2016, which claims the benefit ofU.S. Provisional Application No. 62/271,474, filed Dec. 28, 2015, whichare incorporated herein by reference in their entirety; including anydrawings and sequence listings.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“NKp46-7-PCT_ST25.bd”, created Dec. 19, 2016, which is 376 KB in size.The information in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Immunoglobulin variable regions, and proteins such as antibodies andmultispecific proteins that comprise the variable regions are provided.The proteins can bind and specifically redirect NK cells to lyse atarget cell of interest. The proteins have utility in the treatment ofdisease, notably cancer or infectious disease.

BACKGROUND

Bispecific antibodies binding two different epitopes offer opportunitiesfor increasing specificity, broadening potency, and utilizing novelmechanisms of action that cannot be achieved with a traditionalmonoclonal antibody. A variety of formats for bispecific antibodies thatbind to two targets simultaneously have been reported. Cross-linking twodifferent receptors using a bispecific antibody to inhibit a signalingpathway has shown utility in a number of applications (see, e.g.,Jackman, et al., (2010) J. Biol. Chem. 285:20850-20859). Bispecificantibodies have also been used to neutralize two different receptors. Inother approaches, bispecific antibodies have been used to recruit immuneeffector cells, where T-cell activation is achieved in proximity totumor cells by the bispecific antibody which binds receptorssimultaneously on the two different cell types (see Baeuerle, P. A., etal, (2009) Cancer Res 69(12):4941-4). Approaches developed to date haveprimarily involved bispecific antibodies that link the CD3 complex on Tcells to a tumor-associated antigen. However in other examples,bispecific antibodies having one arm which binds CD16 (FcγRIIIa) andanother which bound to an antigen of interest such as CD19 have beendeveloped (see Kellner et al. (2011) Cancer Lett. 303(2): 128-139).

Natural killer (NK) cells are a subpopulation of lymphocytes that areinvolved in non-conventional immunity. NK cells provide an efficientimmunosurveillance mechanism by which undesired cells such as tumor orvirally-infected cells can be eliminated. Characteristics and biologicalproperties of NK cells include the expression of surface antigensincluding CD16, CD56 and/or CD57, the absence of the alpha/beta orgamma/delta TCR complex on the cell surface; the ability to bind to andkill cells that fail to express “self” MHC/HLA antigens by theactivation of specific cytolytic enzymes, the ability to kill tumorcells or other diseased cells that express a ligand for NK activatingreceptors, and the ability to release protein molecules called cytokinesthat stimulate or inhibit the immune response.

NK cell activity is regulated by a complex mechanism that involves bothactivating and inhibitory signals. Several distinct NK cell receptorshave been identified that play an important role in the NK cell mediatedrecognition and killing of HLA Class I deficient target cells. Onereceptor, although not specific to NK cells, is FcγR3a (CD16) which isresponsible for NK cell mediated ADCC. Another NK cell receptor isNKp46, a member of the Ig superfamily. It is specific to NK cells andits cross-linking, induced by specific mAbs, leads to a strong NK cellactivation resulting in increased intracellular Ca⁺⁺ levels, triggeringof cytotoxicity, and lymphokine release. International patentpublication number WO2005/105858 (Innate Pharma) discloses use ofmonospecific full-length IgG anti-NKp46 antibodies that bind Fcγreceptors for treating hematological malignancies that are Fey-positive.Fc gamma receptors on tumor cells (e.g. B cell malignancies) wereproposed to interact with the Fc domain of the anti-NKp46 antibodieswhich bound NK cells, such that the activated NK cells are brought intoclose proximity with their target cells via the two reactive portions ofthe antibody (e.g. the antigen-recognizing domain and the Fc domain),thereby enhancing the efficiency of the treatment.

To date, no NK cell specific bispecific antibodies have been developed.The depleting agents that recruit NK cytotoxicity such as anti-tumorantibodies are typically full-length IgG1 that mediate ADCC via CD16.Despite the existence of a variety of formats for bispecific antibodies,there remains a need in the art for proteins with new and well-definedmechanisms of action that can provide benefits over and can be used inaddition to full-length antibodies.

SUMMARY OF THE INVENTION

In one aspect, the present invention arises from the discovery ofantibody hypervariable regions that cross-react with both a human NKp46polypeptide and a non-human primate (e.g. e.g. a Macaca fascicularis)NKp46 polypeptide. In another aspect, the present invention arises fromthe discovery of antibody hypervariable regions that remain functionalin in single chain proteins and multi-specific proteins (e.g. apolypeptide, a single chain protein, a multi-chain protein, includingbut not limited to antibody-based protein formats) that bind to NKp46 onNK cells.

Provided also are the epitopes bound by the antigen binding domains. Theantigen domains bind to epitopes on NKp46 that provide for highly potentantigen binding proteins. The epitopes are furthermore shared by humanand non-human primates, including on NKp46 as expressed at the surfaceof an NK cell.

The variable regions, when incorporated into a polypeptide (e.g.antibody, Fc protein, scFv, etc.) that binds NKp46 in monovalent manner,permit the binding to NKp46 on isolated NK cells without triggeringNKp46 activation (in the absence of target cells). The variable regionsare capable of binding NKp46 with high affinity as single chainpolypeptides, e.g., as tandem variable regions separated by a peptidelinker, as well as in F(ab) form, and are capable, as monovalentbinders, to bind to NKp46 epitopes that enable NK cells to be directedto lyse target cells. Such properties make them advantageous in avariety of applications, including in particular, in multispecific (e.g.bispecific) proteins that bind to NKp46 and an antigen of interest viathe heavy and light chain anti-NKp46 variable regions, and to an antigenof interest on a target cell via a hypervariable region (e.g. a heavyand light chain variable region) that specifically binds the antigen ofinterest. Such a multispecific protein is capable of redirecting NKcells to lyse a target cell that expresses the antigen of interest, e.g.a cell that contributes to disease. It will be appreciated that whilethe hypervariable regions and humanized variable regions disclosedherein retain monovalent binding in single chain form and can be usedadvantageously in configurations where they are placed on a singlepolypeptide chain, they can also be used in other applications, such asother proteins where variable regions are on separate chains, e.g.bispecific antibodies and Fc proteins, or more generally monospecificand/or conventional anti-NKp46 antibodies, for example to bind, modulateand/or detect human and/or non-human primate NKp46 polypeptides, in vivoor in vitro (e.g. in a biological sample).

In one aspect, provided is an antigen binding domain (ABD) that bindsboth a human and a non-human primate NKp46 polypeptide (e.g. withsimilar affinity, as assessed for example by surface plasmon resonanceand/or by flow cytometry to NKp46-expression cells, by the methodsherein). In one embodiment, the ABD is capable of binding to a NKp46polypeptide as a single chain antigen binding domain (e.g. an scFv). Inone embodiment, the ABD comprises (or is comprised in) an immunoglobulinheavy and light variable region, e.g. wherein the heavy and light chainvariable domains are placed on a single polypeptide chain. In anotherembodiment heavy and light chain variable domains are placed ondifferent polypeptide chains within a multimeric protein. The heavychain variable region comprises a heavy chain framework region of humanorigin and the light chain variable region comprises a light chainframework regions of human origin, optionally wherein the heavy and/orlight chain framework regions comprise one or more amino acidmodifications (e.g. substitution(s), back-mutation(s) in which a residueis substituted by the residue present in the parental (e.g., murine)framework at the position), optionally wherein the amino acidmodifications provides or increases binding to a non-human primateNKp46. In one embodiment, provided is a protein, an Fc protein, anantibody or an antibody fragment that comprises such an ABD and/orvariable regions. In one aspect, provided is an isolated and/orrecombinant nucleic acid that encodes such an antigen binding domain(ABD).

Examples of such proteins or polypeptides include, e.g. a singlepolypeptide chain NKp46-binding domain (an ABD that binds human NKp46placed on a single polypeptide chain, e.g., a polypeptide comprising aVH and VL of the separated by a (poly)peptide linker). In oneembodiment, the single chain NKp46 binding domain comprises a VH and aVL domain disclosed herein, separated by a peptide linker. The singlepolypeptide chain may be comprised in a multi-chain polypeptide thatcomprises one or more further polypeptide chains, or may be isolated asa single polypeptide chain. Another example of a protein or polypeptidethat comprises an anti-NKp46 ABD is a multimeric protein comprising afirst and a second polypeptide chain, wherein one chain comprises a VHdomain of an anti-NKp46 ABD disclosed herein and the other chaincomprises the VL of an anti-NKp46 ABD disclosed herein, wherein thechains are configured so that the VH and VL form an antigen bindingdomain that binds NKp46.

In one aspect, provided is a protein or polypeptide (e.g. amonospecific, bispecific, or multispecific antibody or protein, a scFv,a F(ab) or F(ab)₂, a multispecific Fc protein) that binds a human NKp46polypeptide, optionally that further bind a non-human primate NKp46polypeptide, wherein the protein nor polypeptide comprises: (a) a heavychain variable region comprising a human heavy chain framework region(FR1, FR2, and FR3) derived from a human IGHV1-69 gene, and heavy chainCDR1, 2 and 3 of a NKp46-1 antibody; and (b) a light chain variableregion comprising a human light chain framework region (FR1, FR2, andFR3) derived from a human IGKV1-33 gene, and light chain CDR1, 2 and 3of a NKp46-1 antibody. Optionally, CDRs as defined by Kabat, Chotia orIMGT numbering. In one embodiment, the protein or polypeptide binds awild-type NKp46 polypeptide but has decreased binding to a mutant NKp46polypeptide having a mutation (e.g., an alanine substitution) any one ormore of the residues K41, E42, E119, Y121 and/or Y194 (with reference toSEQ ID NO:1) compared to binding to the wild-type NKp46). In oneembodiment, the heavy chain variable region comprises an amino acidsequence sharing at least 70%, 80%, 90%, 95% or 98% identity with theamino acid sequence of SEQ ID NO: 3, and/or the light chain variableregion comprises an amino acid sequence sharing at least 70%, 80%, 90%,95% or 98% identity with the amino acid sequence of SEQ ID NO: 4. In oneembodiment, the heavy chain variable region comprises an amino acidsequence of the NKp46-1 H1 or H3 variable domain (or an amino acidsequence sharing at least 80, 90%, 95% or 98% identity therewith) andthe light chain variable region comprises an amino acid sequence of theNKp46-1 L1 variable domain (or an amino acid sequence sharing at least80, 90%, 95% or 98% identity therewith).

In one aspect, provided is a protein or polypeptide (e.g. amonospecific, bispecific, or multispecific antibody or protein, a scFv,a F(ab) or F(ab)₂, a multispecific Fc protein) that binds a human NKp46polypeptide, optionally that further bind a non-human primate NKp46polypeptide, wherein the protein nor polypeptide comprises: (a) a heavychain variable region comprising a human heavy chain framework region(FR1, FR2, and FR3) derived from a IGHV4-30-4 gene, and heavy chainCDR1, 2 and 3 of a NKp46-2 antibody; and (b) a light chain variableregion comprising a human light chain framework region (FR1, FR2, andFR3) derived from a IGKV1-39 gene, and light chain CDR1, 2 and 3 of aNKp46-2 antibody. Optionally, CDRs as defined by Kabat, Chotia or IMGTnumbering. In one embodiment, the heavy chain variable region comprisesan amino acid sequence sharing at least 70%, 80%, 90%, 95% or 98%identity with the amino acid sequence of SEQ ID NO: 5, and/or the lightchain variable region comprises an amino acid sequence sharing at 70%,80%, 90%, 95% or 98% identity with the amino acid sequence of SEQ ID NO:6. In one embodiment, the heavy chain variable region comprises an aminoacid sequence of the NKp46-2 H1, H2 or H3 variable domain (or an aminoacid sequence sharing at least 80, 90%, 95% or 98% identity therewith)and the light chain variable region comprises an amino acid sequence ofthe NKp46-2 L1 variable domain (or an amino acid sequence sharing atleast 80, 90%, 95% or 98% identity therewith).

In one aspect, provided is a protein or polypeptide (e.g. amonospecific, bispecific, or multispecific antibody or protein, a scFv,a F(ab) or F(ab)₂, a multispecific Fc protein) that binds a human NKp46polypeptide, optionally that further bind a non-human primate NKp46polypeptide, wherein the protein nor polypeptide comprises: (a) a heavychain variable region comprising a human heavy chain framework region(FR1, FR2, and FR3) derived from a IGHV1-69 gene, and heavy chain CDR1,2 and 3 of a NKp46-3 antibody; and (b) a light chain variable regioncomprising a human light chain framework region (FR1, FR2, and FR3)derived from a IGKV3-11 and/or a IGKV3-15 gene (e.g. a mosaic variableregion comprising both IGKV3-11 and IGKV3-15 sequences or segments), andlight chain CDR1, 2 and 3 of a NKp46-3 antibody. Optionally, CDRs asdefined by Kabat, Chotia or IMGT numbering. In one embodiment, theprotein or polypeptide binds a wild-type NKp46 polypeptide but hasdecreased binding to a mutant NKp46 polypeptide having a mutation (e.g.,an alanine substitution) any one or more of the residues P132, E133,1135, and/or S136 (with reference to SEQ ID NO:1) compared to binding tothe wild-type NKp46). In one embodiment, the heavy chain variable regioncomprises an amino acid sequence sharing at least 70%, 80%, 90%, 95% or98% identity with the amino acid sequence of SEQ ID NO: 7, and/or thelight chain variable region comprises an amino acid sequence sharing atleast 70%, 80%, 90%, 95% or 98% identity with the amino acid sequence ofSEQ ID NO: 8. In one embodiment, the heavy chain variable regioncomprises an amino acid sequence of the NKp46-3 H1, H3 or H4 variabledomain (or an amino acid sequence sharing at least 80, 90%, 95% or 98%identity therewith) and the light chain variable region comprises anamino acid sequence of the NKp46-3 L1 variable domain (or an amino acidsequence sharing at least 80, 90%, 95% or 98% identity therewith).

In one aspect, provided is a protein or polypeptide (e.g. amonospecific, bispecific, or multispecific antibody or protein, a scFv,a F(ab) or F(ab)₂, a multispecific Fc protein) that binds a human NKp46polypeptide, optionally that further bind a non-human primate NKp46polypeptide, wherein the protein nor polypeptide comprises: (a) a heavychain variable region comprising a human heavy chain framework region(FR1, FR2, and FR3) derived from a IGHV1-46 and/or a IGHV1-69 gene (e.g.a mosaic variable region comprising both IGHV1-46 and IGHV1-69 sequencesor segments), and heavy chain CDR1, 2 and 3 of a NKp46-4 antibody; and(b) a light chain variable region comprising a human light chainframework region (FR1, FR2, and FR3) derived from a IGKV1-NL1 gene, andlight chain CDR1, 2 and 3 of a NKp46-4 antibody. Optionally, CDRs asdefined by Kabat, Chotia or IMGT numbering. In one embodiment, theprotein or polypeptide binds a wild-type NKp46 polypeptide but hasdecreased binding to a mutant NKp46 polypeptide having a mutation (e.g.,an alanine substitution) any one or more of the residues R101, V102,E104 and/or L105 (with reference to SEQ ID NO:1) compared to binding tothe wild-type NKp46). In one embodiment, the heavy chain variable regioncomprises an amino acid sequence sharing at least 70%, 80%, 90%, 95% or98% identity with the amino acid sequence of SEQ ID NO: 9, and/or thelight chain variable region comprises an amino acid sequence sharing atleast 70%, 80%, 90%, 95% or 98% identity with the amino acid sequence ofSEQ ID NO: 10. In one embodiment, the heavy chain variable regioncomprises an amino acid sequence of the NKp46-4 H1 variable domain (oran amino acid sequence sharing at least 80, 90%, 95% or 98% identitytherewith) and the light chain variable region comprises an amino acidsequence of the NKp46-4 L2 variable domain (or an amino acid sequencesharing at least 80, 90%, 95% or 98% identity therewith).

In one aspect, provided is a protein or polypeptide (e.g. amonospecific, bispecific, or multispecific antibody or protein, a scFv,a F(ab) or F(ab)₂, a multispecific Fc protein) that binds a human NKp46polypeptide, optionally that further bind a non-human primate NKp46polypeptide, wherein the protein nor polypeptide comprises: (a) a heavychain variable region comprising a human heavy chain framework region(FR1, FR2, FR3) derived from a IGHV4-30-4 gene, and heavy chain CDR1, 2and 3 of a NKp46-9 antibody; and (b) a light chain variable regioncomprising a human light chain framework region (FR1, FR2, FR3) derivedfrom a IGKV1-39 gene, and light chain CDR1, 2 and 3 of a NKp46-9antibody. Optionally, CDRs as defined by Kabat, Chotia or IMGTnumbering. In one embodiment, the heavy chain variable region comprisesan amino acid sequence sharing at least 70%, 80%, 90%, 95% or 98%identity with the amino acid sequence of SEQ ID NO: 13, and/or the lightchain variable region comprises an amino acid sequence sharing at least70%, 80%, 90%, 95% or 98% identity with the amino acid sequence of SEQID NO: 14. In one embodiment, the heavy chain variable region comprisesan amino acid sequence of the NKp46-9 H2 variable domain (or an aminoacid sequence sharing at least 80, 90%, 95% or 98% identity therewith)and the light chain variable region comprises an amino acid sequence ofthe NKp46-9 L1 or L2 variable domain (or an amino acid sequence sharingat least 80, 90%, 95% or 98% identity therewith). In one embodiment, theheavy chain variable region comprises an amino acid sequence of theNKp46-9 H3 variable domain (or an amino acid sequence sharing at least80, 90%, 95% or 98% identity therewith) and the light chain variableregion comprises an amino acid sequence of the NKp46-9 L1 or L2 variabledomain (or an amino acid sequence sharing at least 80, 90%, 95% or 98%identity therewith).

In one aspect, provided is an antigen binding domain comprising a heavychain variable region (VH) and a light chain variable region (VL) eachcontaining human FR1, 2 and 3 framework regions, or a protein orpolypeptide comprising such ABD (e.g. a monoclonal antibody, an scFv, amultispecific polypeptide, a bispecific antibody, a DART® or BiTe® orprotein comprising such, etc.) that binds a human and a non-humanprimate NKp46 polypeptide, e.g. a cell surface NKp46 polypeptide,wherein the protein or polypeptide comprises a VH and VL combinationselected from the group consisting of:

-   -   (a) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% (or 100%) identical to the amino acid sequence        of the NKp46-1 H1 or H3 variable domain shown in SEQ ID NOS: 199        or 200, respectively, and a VL comprising an amino acid sequence        at least 70%, 80%, 90%, 95% or 98% identical to the amino acid        sequence of the NKp46-1 L1 variable domain shown in SEQ ID NO:        201;    -   (b) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% (or 100%) identical to the amino acid sequence        of the NKp46-2 H1, H2 or H3 variable domain shown in SEQ ID NOS:        202, 203 or 203, respectively, and a VL comprising an amino acid        sequence at least 70%, 80%, 90%, 95% or 98% identical to the        amino acid sequence of the NKp46-2 L1 variable domain shown in        SEQ ID NO: 205;    -   (c) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% (or 100%) identical to the amino acid sequence        of the NKp46-3 H1, H3 or H4 variable domain shown in SEQ ID NOS:        206, 207 or 208, respectively, and a VL comprising an amino acid        sequence at least 70%, 80%, 90%, 95% or 98% identical to the        amino acid sequence of the NKp46-3 L1 variable domain shown in        SEQ ID NO: 209;    -   (d) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% (or 100%) identical to the amino acid sequence        of the NKp46-4 H1, H2 or H3 variable domain shown in SEQ ID NOS:        210, 211 or 212, and a VL comprising an amino acid sequence at        least 70%, 80%, 90%, 95% or 98% identical to the amino acid        sequence of the NKp46-4 L2 variable domain shown in SEQ ID NO:        213;    -   (e) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% (or 100%) identical to the amino acid sequence        of the NKp46-9 H2 variable domain shown in SEQ ID NO: 215, and a        VL comprising an amino acid sequence at least 70%, 80%, 90%, 95%        or 98% identical to the amino acid sequence of the NKp46-9 L1 or        L2 variable domain shown in SEQ ID NOS: 217 or 218,        respectively; or    -   (f) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% (or 100%) identical to the amino acid sequence        of the NKp46-9 H3 variable domain shown in SEQ ID NO: 215, and a        VL comprising an amino acid sequence at least 70%, 80%, 90%, 95%        or 98% identical to the amino acid sequence of the NKp46-9 L1 or        L2 variable domain shown in SEQ ID NOS: 217 or 218,        respectively.

In one aspect, provided is an antigen binding domain comprising a heavychain variable region (VH) and a light chain variable region (VL) eachcontaining human FR1, 2 and 3 framework regions, or a protein orpolypeptide comprising such ABD that binds a human and a non-humanprimate NKp46 polypeptide, wherein the protein or polypeptide comprisesa VH and VL combination selected from the group consisting of:

-   -   (a) a VH comprising a CDR1, 2 and 3 of the VH domain having the        amino acid sequence shown in SEQ ID NO: 3 and a FR1, 2 and 3 of        a human IGHV1-69 gene segment, and a VL comprising a CDR1, 2 and        3 of the VL domain having the amino acid sequence shown in SEQ        ID NO: 4 and a FR1, 2 and 3 of a human IGKV1-33 gene segment;    -   (b) a VH comprising a CDR1, 2 and 3 of the VH domain having the        amino acid sequence shown in SEQ ID NO: 5 and a FR1, 2 and 3 of        a human IGHV4-30-4 gene segment, and a VL comprising a CDR1, 2        and 3 of the VL domain having the amino acid sequence shown in        SEQ ID NO: 6 and a FR1, 2 and 3 of a human IGKV1-39 gene        segment;    -   (c) a VH comprising a CDR1, 2 and 3 of the VH domain having the        amino acid sequence shown in SEQ ID NO: 7 and a FR1, 2 and 3 of        a human IGHV1-69 gene segment, and a VL comprising a CDR1, 2 and        3 of the VL domain having the amino acid sequence shown in SEQ        ID NO: 8 and a FR1, 2 and 3 of a human IGKV3-11 and/or IGKV3-15        gene segment;    -   (d) a VH comprising a CDR1, 2 and 3 of the VH domain having the        amino acid sequence shown in SEQ ID NO: 9 and a FR1, 2 and 3 of        a human IGHV1-46 and/or a IGHV1-69 gene segment, and a VL        comprising a CDR1, 2 and 3 of the VL domain having the amino        acid sequence shown in SEQ ID NO: 10 and a FR1, 2 and 3 of a        human IGKV1-NL1 gene segment;    -   (e) a VH comprising a CDR1, 2 and 3 of the VH domain having the        amino acid sequence shown in SEQ ID NO: 13 and a FR1, 2 and 3 of        a human IGHV4-30-4 gene segment, and a VL comprising a CDR1, 2        and 3 of the VL domain having the amino acid sequence shown in        SEQ ID NO: 14 and a FR1, 2 and 3 of a human IGKV1-39 gene        segment.

In any aspect, an antigen binding domain may comprise a VH and a VLcomprising an amino acid sequence at least 80%, 90%, 95% or 98% (or100%) identical to the respective VH and VL of any one of antibodies:NKp46-1 H1L1, NKp46-1 H3L1, NKp46-2 H1L1, NKp46-2 H2L1, NKp46-2 H3L1,NKp46-3 H1L1, NKp46-3 H3L1, NKp46-3 H4L1, NKp46-4 H1L2, NKp46-4 H2L2,NKp46-4 H3L2, NKp46-9 H2L1, NKp46-9 H2L2, NKp46-9 H3L1 or NKp46-9 H3L2.

In one aspect, provided is an antigen binding domain comprising a heavychain variable region (VH) and a light chain variable region (VL) eachcontaining human FR1, 2 and 3 framework regions, or a protein orpolypeptide (e.g., an antibody, multispecific protein) comprising suchABD that binds a NKp46 polypeptide, wherein the protein or polypeptidecomprising an amino acid sequence at least 80%, 90%, 95% or 98% (or100%) identical to the respective VH and VL of any one of antibodies:NKp46-1 H1L1, NKp46-1 H3L1, NKp46-2 H1L1, NKp46-2 H2L1, NKp46-2 H3L1,NKp46-3 H1L1, NKp46-3 H3L1, NKp46-3 H4L1, NKp46-4 H1L2, NKp46-4 H2L2,NKp46-4 H3L2, NKp46-9 H2L1, NKp46-9 H2L2, NKp46-9 H3L1 or NKp46-9 H3L2.VH and VL sequences of these antibodies are listed in Table C andExample 1 part B.

In one embodiment, a NKp46-1 VL can comprising an amino acidsubstitution at Kabat position 87, optionally wherein the residue atposition 87 is a phenylalanine. A NKp46-1 VH can comprise an amino acidsubstitution at Kabat residues 27, 66 and/or 67, optionally further asubstitution at Kabat residues 37, 48 and/or 91. Optionally the residuepresent at the particular position is the residue substituted at theposition herein, e.g., as shown in Example 1.

In one embodiment, a NKp46-2 VL can comprise an amino acid substitutionat Kabat position 48, optionally wherein the residue at position 48 is avaline. A NKp46-2 VH can comprise an amino acid substitution at Kabatresidues 27 and/or 71, optionally further a substitution at Kabatresidues 48 and/or 67, optionally further a substitution at Kabatresidue 31. Optionally the residue present at the particular position isthe residue substituted at the position herein, e.g., as shown inExample 1.

In one embodiment, a NKp46-3 VL can comprise an amino acid substitutionat Kabat position 49, optionally wherein the residue at position 49 is alysine. A NKp46-3 VH can comprise an amino acid substitution at Kabatresidue 27, optionally further a substitution at Kabat residues 48and/or 67, optionally further a substitution at Kabat residue 69.Optionally the residue present at the particular position is the residuesubstituted at the position herein, e.g., as shown in Example 1.

In one embodiment, a NKp46-4 VL can comprise an amino acid substitutionat Kabat position 36 and/or 48, optionally wherein the residue atposition 36 is a phenylalanine, optionally wherein the residue atposition 48 is a valine. A NKp46-4 VH can comprise an amino acidsubstitution at Kabat residues 30, 48 and/or 93, optionally further asubstitution at Kabat residue 67, optionally further a substitution atKabat residue 69. Optionally the residue present at the particularposition is the residue substituted at the position herein, e.g., asshown in Example 1.

In one embodiment, a NKp46-9 VL can comprise an amino acid substitutionat Kabat position 36, optionally wherein the residue at position 36 is acysteine, optionally further a substitution at Kabat residue 48,optionally wherein the residue at position 48 is a valine. A NKp46-9 VHcan comprise an amino acid substitution at Kabat residue 71, optionallyfurther a substitution at Kabat residue 27, optionally further asubstitution at Kabat residue 48 and/or 67. Optionally the residuepresent at the particular position is the residue substituted at theposition herein, e.g., as shown in Example 1.

In one aspect of any embodiment, the protein or polypeptide binds NKp46in monovalent manner. In aspect of any embodiment, the protein orpolypeptide comprises or contains no more than one antigen bindingdomain that binds NKp46.

In one embodiment, the protein or polypeptide comprises a human Fcdomain or a portion thereof, wherein the Fc domain or portion thereof iscapable of binding to human neonatal Fc receptor (FcRn) and to humanCD16 polypeptide. Because FcγRIIIa (CD16) is not present on all NKcells, conventional therapeutic antibodies (e.g. of human isotypes IgG1)designed to exert antibody-dependent cellular toxicity (ADCC) viaFcγRIIIa may not mobilize all NK cells; the present proteins on theother hand enable all NK cells to be solicited via NKp46, and theproteins will thus be useful to activate or increase the cytolyticactivity of NKp46⁺CD16⁻ NK cells as well as NKp46⁺CD16⁺ NK cells.

In one embodiment, the protein or polypeptide comprises a human Fcdomain or a portion thereof, wherein the Fc domain or a portion thereofis capable of binding to human neonatal Fc receptor (FcRn) and lacksbinding to human CD16 polypeptide.

In one embodiment, the protein or polypeptide is a single chainpolypeptide. In one embodiment, the protein or polypeptide is amultimeric polypeptide, optionally a dimeric, trimeric or tetramericprotein.

Advantageously, in one embodiment, the presence of NK cells and targetcells, a multi-specific protein can bind (i) to antigen of interest ontarget cells and (ii) to NKp46 on NK cells, and, when bound to bothantigen of interest on target cells and NKp46, can induce signaling inand/or activation of the NK cells through NKp46 (the protein acts as anNKp46 agonist), thereby promoting activation of NK cells and/or lysis oftarget cells, notably via the activating signal transmitted by NKp46. Inspecific advantageous embodiments, the multi-specific comprises a singleantigen binding domain that binds NKp46 (the protein binds to NKp46 inmonovalent fashion). In one embodiment the protein is capable of, whenbound to both antigen of interest on target cells and NKp46 on NK cells,inducing signaling in the NK cells through NKp46. In one embodiment, theprotein comprises a first antigen binding domain and a second antigenbinding domain, wherein one of the first or second antigen bindingdomains binds to a human NKp46 polypeptide and the other of the first orsecond antigen binding domains binds an antigen of interest expressed ona target cell.

In one embodiment, a multispecific protein is monomeric. In oneembodiment a multispecific protein is a dimer, e.g. a heterodimer,trimer or tetramer. In one embodiment, the protein is a heterodimer,heterotrimer or a tetramer comprising a first polypeptide chaincomprising a VH and a VL domain of an ABD that binds NKp46 (a VH and VLdisclosed herein) separated by a linker peptide, and optionally an Fcdomain, and a second polypeptide chain comprising one or more variableregions or antigen binding domains that bind an antigen of interest, andoptionally an Fc domain. In one embodiment, both a first and a secondpolypeptide chain comprise an Fc domain, and the protein comprises adimeric Fc domain, optionally wherein the Fc domain is capable ofbinding to human CD16. In one embodiment, the monomeric or dimericprotein comprises a protein with a domain structure in which an Fcdomain is interposed between the antigen binding domain (ABD) that bindsto NKp46 and the antigen binding domain that binds an antigen ofinterest. In one embodiment the multispecific Fc-derived polypeptide isa bispecific antibody.

In one embodiment of any of the protein herein, the antigen bindingdomain of a multispecific protein that binds to an antigen of interestbinds to an antigen (e.g. polypeptide) expressed by a target cell whichsought to be lysed by an NK cell. Optionally such an antigen isexpressed by a cancer cell, a tumor associated immune cell (e.g. animmune suppressor or regulatory cell), a virally infected cell, or acell that contributes to an autoimmunity or inflammatory disease.Optionally, the antigen of interest is a cancer antigen known to becapable of undergoing intracellular internalization when contacted witha full length human IgG1 antibody that binds specifically thereto.

In one aspect of any of the embodiments herein, provided is arecombinant nucleic acid encoding a VH and/or a VL, an antibody heavyand/or light chain, a single chain antigen binding domain, a first,second, third or further polypeptide chain of any of the proteins of thedisclosure. In one aspect of any of the embodiments herein, provided isa recombinant host cell comprising a nucleic acid of the disclosure.

Any of the methods can further be characterized as comprising any stepdescribed in the application, including notably in the “DetailedDescription of the Invention”). The invention further relates to methodsof identifying, testing and/or making proteins described herein. Theinvention further relates to a multispecific protein obtainable by anyof present methods. The disclosure further relates to pharmaceutical ordiagnostic formulations of the multispecific protein disclosed herein.The disclosure further relates to methods of using the multispecificprotein in methods of treatment or diagnosis.

These and additional advantageous aspects and features of the inventionmay be further described elsewhere herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that Anti-CD19-F1-Anti-CD3 does not cause T/B cellaggregation in the presence of B221 (CD19) or JURKAT (CD3) cell lineswhen separate, but it does cause aggregation of cells when both B221 andJURKAT cells are co-incubated.

FIGS. 2A to 2F show different domain arrangements of bispecific proteinsproduced.

FIGS. 3A and 3B show respectively bispecific F1 and F2 antibodies havingNKp46 binding region based on NKp46-1, NKp46-2, NKp46-3 or NKp46-4 areable to direct resting NK cells to their CD19-positive Daudi tumortarget cells, while isotype control antibody did not lead to eliminationof the Daudi cells. Rituximab (RTX) served as positive control of ADCC,where the maximal response obtained with RTX (at 10 μg/ml in this assay)was 21.6% specific lysis.

FIG. 4A (top panel CD107, bottom panel CD69) shows bispecific antibodieshaving NKp46 and CD19 binding regions in an F2 format protein do notactivate resting NK cells in the absence of target cells, however fulllength anti-NKp46 antibodies as well as positive control alemtuzumab didactivate NK cells. FIG. 4B shows that in presence of Daudi target cells,bispecific anti-NKp46×anti-CD19 antibodies (including each of theNKp46-1, NKp46-2, NKp46-3 or NKp46-4 binding domains) activated restingNK cells (top panel CD107, bottom panel CD69), while full-lengthanti-CD19 showed at best only very low activation of NK cells. Neitherfull-length anti-NKp46 antibodies or alemtuzumab showed substantialincrease in activation beyond what was observed in presence of NK cellsalone. FIG. 4C (top panel CD107, bottom panel CD69) shows that in thepresence of CD19-negative HUT78 cells, none of the bispecificanti-NKp46×anti-CD19 antibody (including each of the NKp46-1, NKp46-2,NKp46-3 or NKp46-4 variable regions) activated NK cells. However, thefull-length anti-NKp46 antibodies and alemtuzumab caused detectableactivation of NK cells at a similar level observed in presence of NKcells alone. Isotype control antibody did not induce activation.

FIGS. 5A and 5B show that at low effector:target ratio of 1:1 each ofthe bispecific anti-NKp46×anti-CD19 antibody activated NK cells in thepresence of Daudi cells, and that bispecific anti-NKp46×anti-CD19 werefar more potent than the anti-CD19 antibody as a full-length human IgG1as ADCC inducing antibody. Top panel is CD107 (FIG. 5A) and bottom panelshows CD69 (FIG. 5B).

FIGS. 6A and 6B show that each NKp46×CD19 bispecific protein (singlechain format F3, and multimeric formats F5 and F6) induced specificlysis of Daudi (FIG. 6A) or B221 (FIG. 6B) cells by human KHYG-1CD16-negative hNKp46-positive NK cell line, while rituximab and humanIgG1 isotype control (IC) antibodies did not.

FIG. 7 shows the NKp46×CD19 bispecific protein in F5 format whose Fcdomain binds CD16 is far more potent in mediating Daudi target celllysis that the full-length IgG1 anti-CD19 antibody or the F6 formatbispecific protein. The bispecific anti-CD19 in F6 format whose Fcdomain does not bind CD16 was as potent in mediating NK cell lysis ofDaudi target cells as the full-length IgG1 anti-CD19 antibody, which isremarkable considering that the control IgG1 anti-CD19 antibody bindsCD19 bivalently.

DETAILED DESCRIPTION OF THE INVENTION

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one.

Where “comprising” is used, this can optionally be replaced by“consisting essentially of”, more optionally by “consisting of”.

As used herein, the term “antigen binding domain” refers to a domaincomprising a three-dimensional structure capable of immunospecificallybinding to an epitope. Thus, in one embodiment, said domain can comprisea hypervariable region, optionally a VH and/or VL domain of an antibodychain, optionally at least a VH domain. In another embodiment, thebinding domain may comprise at least one complementarity determiningregion (CDR) of an antibody chain. In another embodiment, the bindingdomain may comprise a polypeptide domain from a non-immunoglobulinscaffold.

The term “antibody” herein is used in the broadest sense andspecifically includes full-length monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments and derivatives, so long as they exhibit the desiredbiological activity. Various techniques relevant to the production ofantibodies are provided in, e.g., Harlow, et al., ANTIBODIES: ALABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., (1988). An “antibody fragment” comprises a portion of afull-length antibody, e.g. antigen-binding or variable regions thereof.Examples of antibody fragments include Fab, Fab′, F(ab)₂, F(ab)₂,F(ab)₃, Fv (typically the VL and VH domains of a single arm of anantibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VHand CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VhH,and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, andkappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10: 949-57);camel IgG; IgNAR; and multispecific antibody fragments formed fromantibody fragments, and one or more isolated CDRs or a functionalparatope, where isolated CDRs or antigen-binding residues orpolypeptides can be associated or linked together so as to form afunctional antibody fragment. Various types of antibody fragments havebeen described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol2005; 23, 1126-1136; WO2005040219, and published U.S. PatentApplications 20050238646 and 20020161201.

The term “antibody derivative”, as used herein, comprises a full-lengthantibody or a fragment of an antibody, e.g. comprising at leastantigen-binding or variable regions thereof, wherein one or more of theamino acids are chemically modified, e.g., by alkylation, PEGylation,acylation, ester formation or amide formation or the like. Thisincludes, but is not limited to, PEGylated antibodies,cysteine-PEGylated antibodies, and variants thereof.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity-determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain;Kabat et al. 1991) and/or those residues from a “hypervariable loop”(e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in thisregion is performed by the method described in Kabat et al., supra.Phrases such as “Kabat position”, “variable domain residue numbering asin Kabat” and “according to Kabat” herein refer to this numbering systemfor heavy chain variable domains or light chain variable domains. Usingthe Kabat numbering system, the actual linear amino acid sequence of apeptide may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or CDR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of CDR H2and inserted residues (e.g. residues 82a, 82b, and 82c, etc. accordingto Kabat) after heavy chain FR residue 82. The Kabat numbering ofresidues may be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

By “framework” or “FR” residues as used herein is meant the region of anantibody variable domain exclusive of those regions defined as CDRs.Each antibody variable domain framework can be further subdivided intothe contiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4).

By “constant region” as defined herein is meant an antibody-derivedconstant region that is encoded by one of the light or heavy chainimmunoglobulin constant region genes. By “constant light chain” or“light chain constant region” as used herein is meant the region of anantibody encoded by the kappa (Ckappa) or lambda (Clambda) light chains.The constant light chain typically comprises a single domain, and asdefined herein refers to positions 108-214 of Ckappa, or Clambda,wherein numbering is according to the EU index (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda). By“constant heavy chain” or “heavy chain constant region” as used hereinis meant the region of an antibody encoded by the mu, delta, gamma,alpha, or epsilon genes to define the antibody's isotype as IgM, IgD,IgG, IgA, or IgE, respectively. For full length IgG antibodies, theconstant heavy chain, as defined herein, refers to the N-terminus of theCH1 domain to the C-terminus of the CH3 domain, thus comprisingpositions 118-447, wherein numbering is according to the EU index.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of apolypeptide, multispecific polypeptide or ABD, or any other embodimentsas outlined herein.

By “single-chain Fv” or “scFv” as used herein are meant antibodyfragments comprising the VH and VL domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. Methods for producing scFvs are well known in the art. For areview of methods for producing scFvs see Pluckthun in The Pharmacologyof Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994).

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domains Cγ2(CH2) and Cγ3 (CH3) and the hinge between Cγ1 and Cγ2. Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues at least C226, P230 orA231 to its carboxyl-terminus, wherein the numbering is according to theEU index. Fc may refer to this region in isolation, or this region inthe context of an Fc polypeptide, as described below. By “Fcpolypeptide” or “Fc-derived polypeptide” as used herein is meant apolypeptide that comprises all or part of an Fc region. Fc polypeptidesinclude but is not limited to antibodies, Fc fusions and Fc fragments.

By “variable region” as used herein is meant the region of an antibodythat comprises one or more Ig domains substantially encoded by any ofthe VL (including Vkappa (VK) and Vlambda) and/or VH genes that make upthe light chain (including kappa and lambda) and heavy chainimmunoglobulin genetic loci respectively. A light or heavy chainvariable region (VL or VH) consists of a “framework” or “FR” regioninterrupted by three hypervariable regions referred to as“complementarity determining regions” or “CDRs”. The extent of theframework region and CDRs have been precisely defined, for example as inKabat (see “Sequences of Proteins of Immunological Interest,” E. Kabatet al., U.S. Department of Health and Human Services, (1983)), and as inChothia. The framework regions of an antibody, that is the combinedframework regions of the constituent light and heavy chains, serves toposition and align the CDRs, which are primarily responsible for bindingto an antigen.

The term “specifically binds to” means that an antibody or polypeptidecan bind preferably in a competitive binding assay to the bindingpartner, e.g. NKp46, as assessed using either recombinant forms of theproteins, epitopes therein, or native proteins present on the surface ofisolated target cells. Competitive binding assays and other methods fordetermining specific binding are further described below and are wellknown in the art.

When an antibody or polypeptide is said to “compete with” a particularmonoclonal antibody (e.g. NKp46-1, -2, -4, -6 or -9 in the context of ananti-NKp46 mono- or bi-specific antibody), it means that the antibody orpolypeptide competes with the monoclonal antibody in a binding assayusing either recombinant target (e.g. NKp46) molecules or surfaceexpressed target (e.g. NKp46) molecules. For example, if a test antibodyreduces the binding of NKp46-1, -2, -4, -6 or -9 to a NKp46 polypeptideor NKp46-expressing cell in a binding assay, the antibody is said to“compete” respectively with NKp46-1, -2, -4, -6 or -9.

The term “affinity”, as used herein, means the strength of the bindingof an antibody or polypeptide to an epitope. The affinity of an antibodyis given by the dissociation constant K_(D), defined as[Ab]×[Ag]/[Ab−Ag], where [Ab−Ag] is the molar concentration of theantibody-antigen complex, [Ab] is the molar concentration of the unboundantibody and [Ag] is the molar concentration of the unbound antigen. Theaffinity constant K_(A) is defined by 1/K_(D). Preferred methods fordetermining the affinity of mAbs can be found in Harlow, et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), whichreferences are entirely incorporated herein by reference. One preferredand standard method well known in the art for determining the affinityof mAbs is the use of surface plasmon resonance (SPR) screening (such asby analysis with a BIAcore™ SPR analytical device).

Within the context of this invention a “determinant” designates a siteof interaction or binding on a polypeptide.

The term “epitope” refers to an antigenic determinant, and is the areaor region on an antigen to which an antibody or polypeptide binds. Aprotein epitope may comprise amino acid residues directly involved inthe binding as well as amino acid residues which are effectively blockedby the specific antigen binding antibody or peptide, i.e., amino acidresidues within the “footprint” of the antibody. It is the simplest formor smallest structural area on a complex antigen molecule that cancombine with e.g., an antibody or a receptor. Epitopes can be linear orconformational/structural. The term “linear epitope” is defined as anepitope composed of amino acid residues that are contiguous on thelinear sequence of amino acids (primary structure). The term“conformational or structural epitope” is defined as an epitope composedof amino acid residues that are not all contiguous and thus representseparated parts of the linear sequence of amino acids that are broughtinto proximity to one another by folding of the molecule (secondary,tertiary and/or quaternary structures). A conformational epitope isdependent on the 3-dimensional structure. The term ‘conformational’ istherefore often used interchangeably with ‘structural’.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. An example ofamino acid modification herein is a substitution. By “amino acidmodification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence. By “amino acid substitution”or “substitution” herein is meant the replacement of an amino acid at agiven position in a protein sequence with another amino acid. Forexample, the substitution Y50W refers to a variant of a parentpolypeptide, in which the tyrosine at position 50 is replaced withtryptophan. A “variant” of a polypeptide refers to a polypeptide havingan amino acid sequence that is substantially identical to a referencepolypeptide, typically a native or “parent” polypeptide. The polypeptidevariant may possess one or more amino acid substitutions, deletions,and/or insertions at certain positions within the native amino acidsequence.

“Conservative” amino acid substitutions are those in which an amino acidresidue is replaced with an amino acid residue having a side chain withsimilar physicochemical properties. Families of amino acid residueshaving similar side chains are known in the art, and include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

Preferred methods for determining identity are designed to give thelargest match between the sequences tested. Methods of determiningidentity are described in publicly available computer programs.Preferred computer program methods for determining identity between twosequences include the GCG program package, including GAP (Devereux etal., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX programis publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well knownSmith Waterman algorithm may also be used to determine identity.

An “isolated” molecule is a molecule that is the predominant species inthe composition wherein it is found with respect to the class ofmolecules to which it belongs (i.e., it makes up at least about 50% ofthe type of molecule in the composition and typically will make up atleast about 70%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or more of the species of molecule, e.g.,peptide, in the composition). Commonly, a composition of a polypeptidewill exhibit 98%, 98%, or 99% homogeneity for polypeptides in thecontext of all present peptide species in the composition or at leastwith respect to substantially active peptide species in the context ofproposed use.

In the context herein, “treatment” or “treating” refers to preventing,alleviating, managing, curing or reducing one or more symptoms orclinically relevant manifestations of a disease or disorder, unlesscontradicted by context. For example, “treatment” of a patient in whomno symptoms or clinically relevant manifestations of a disease ordisorder have been identified is preventive or prophylactic therapy,whereas “treatment” of a patient in whom symptoms or clinically relevantmanifestations of a disease or disorder have been identified generallydoes not constitute preventive or prophylactic therapy.

As used herein, “NK cells” refers to a sub-population of lymphocytesthat is involved in non-conventional immunity. NK cells can beidentified by virtue of certain characteristics and biologicalproperties, such as the expression of specific surface antigensincluding CD56 and/or NKp46 for human NK cells, the absence of thealpha/beta or gamma/delta TCR complex on the cell surface, the abilityto bind to and kill cells that fail to express “self” MHC/HLA antigensby the activation of specific cytolytic machinery, the ability to killtumor cells or other diseased cells that express a ligand for NKactivating receptors, and the ability to release protein moleculescalled cytokines that stimulate or inhibit the immune response. Any ofthese characteristics and activities can be used to identify NK cells,using methods well known in the art. Any subpopulation of NK cells willalso be encompassed by the term NK cells. Within the context herein“active” NK cells designate biologically active NK cells, including NKcells having the capacity of lysing target cells or enhancing the immunefunction of other cells. NK cells can be obtained by various techniquesknown in the art, such as isolation from blood samples, cytapheresis,tissue or cell collections, etc. Useful protocols for assays involvingNK cells can be found in Natural Killer Cells Protocols (edited byCampbell K S and Colonna M). Human Press. pp. 219-238 (2000).

The term “internalization”, used interchangeably with “intracellularinternalization”, refers to the molecular, biochemical and cellularevents associated with the process of translocating a molecule from theextracellular surface of a cell to the intracellular surface of a cell.The processes responsible for intracellular internalization of moleculesare well-known and can involve, inter alia, the internalization ofextracellular molecules (such as hormones, antibodies, and small organicmolecules); membrane-associated molecules (such as cell-surfacereceptors); and complexes of membrane-associated molecules bound toextracellular molecules (for example, a ligand bound to a transmembranereceptor or an antibody bound to a membrane-associated molecule). Thus,“inducing and/or increasing internalization” comprises events whereinintracellular internalization is initiated and/or the rate and/or extentof intracellular internalization is increased.

As used herein, an agent that has “agonist” activity at Nkp46 is anagent that can cause or increase “NKp46 signaling”. “Nkp46 signaling”refers to an ability of a NKp46 polypeptide to activate or transduce anintracellular signaling pathway. Changes in NKp46 signaling activity canbe measured, for example, by assays designed to measure changes in NKp46signaling pathways, e.g. by monitoring phosphorylation of signaltransduction components, assays to measure the association of certainsignal transduction components with other proteins or intracellularstructures, or in the biochemical activity of components such askinases, or assays designed to measure expression of reporter genesunder control of NKp46-sensitive promoters and enhancers, or indirectlyby a downstream effect mediated by the NKp46 polypeptide (e.g.activation of specific cytolytic machinery in NK cells). Reporter genescan be naturally occurring genes (e.g. monitoring cytokine production)or they can be genes artificially introduced into a cell. Other genescan be placed under the control of such regulatory elements and thusserve to report the level of NKp46 signaling.

“NKp46” refers to a protein or polypeptide encoded by the Ncr1 gene orby a cDNA prepared from such a gene. Any naturally occurring isoform,allele or variant is encompassed by the term NKp46 polypeptide (e.g., anNKp46 polypeptide 90%, 95%, 98% or 99% identical to SEQ ID NO 1, or acontiguous sequence of at least 20, 30, 50, 100 or 200 amino acidresidues thereof). The 304 amino acid residue sequence of human NKp46(isoform a) is shown as follows:

(SEQ ID NO: 1) MSSTLPALLC VGLCLSQRIS AQQQTLPKPF IWAEPHFMVPKEKQVTICCQ GNYGAVEYQL HFEGSLFAVD RPKPPERINKVKFYIPDMNS RMAGQYSCIY RVGELWSEPS NLLDLVVTEMYDTPTLSVHP GPEVISGEKV TFYCRLDTAT SMFLLLKEGRSSHVQRGYGK VQAEFPLGPV TTAHRGTYRC FGSYNNHAWSFPSEPVKLLV TGDIENTSLA PEDPTFPADT WGTYLLTTETGLQKDHALWD HTAQNLLRMG LAFLVLVALV WFLVEDWLSR KRTRERASRA STWEGRRRLN TQTL.

SEQ ID NO: 1 corresponds to NCBI accession number NP_004820, thedisclosure of which is incorporated herein by reference. The human NKp46mRNA sequence is described in NCBI accession number NM_004829, thedisclosure of which is incorporated herein by reference. The amino acidresidue sequence of the extracellular domain of the Macaca fascicularis(cynomolgus) NKp46 polypeptide is shown as follows:

     MSSTLRALLCLGLCLSQRISAPKQTLPKPIIRAESTYMVPKEKQATLCCQGSYGAVEYQLHFEGSLFAVERPKPPERINGVKFHIPDMNSRKAGRYSCIYRVGELWSERSDLLDLVVTEMYDTPTLSVHPGPEVTSGEKVTFYCRLDTATSMFLLLKEGRSRDVQRSYGKVQAEFPMGPVTTAHRGSYRCFGSYNNYAWSFPSEPVKLLVTGDIENTSLAPTDPTFPDSWDTCLLTRETGLQKDL ALWDHTAQN.Producing Anti-NKp46 Antibodies

The anti-NKp46 antigen binding domains bind an extra-cellular portion ofhuman NKp46 polypeptide. In one aspect, the protein that comprises a VHand VL of the disclosure is a humanized antibody or comprises an antigenbinding fragment thereof. In one aspect, the antibody comprises aconstant domain selected from a human IgG1, IgG2, IgG3 and IgG4 constantdomain, optionally comprising one or more amino acid modifications. Inone aspect, anti-NKp46 antibody comprises an antibody fragment selectedfrom a Fab fragment, a Fab′ fragment, a Fab′-SH fragment, a F(ab)2fragment, a F(ab′)2 fragment, an Fv fragment, a Heavy chain Ig (a llamaor camel Ig), a V_(HH) fragment, a single domain FV, and a single-chainantibody fragment. In one aspect, the anti-NKp46 antibody comprises asynthetic or semisynthetic antibody-derived molecule selected from ascFV, a dsFV, a minibody, a diabody, a triabody, a kappa body, an IgNAR;and a multispecific antibody. The agent can optionally further comprisean Fc domain. In one aspect, the antibody is in at least partiallypurified form. In one aspect, the antibody is in essentially isolatedform.

Antibodies may be produced by a variety of techniques known in the art.Immunological screening assays in which antibody competition can beassessed can be used to select for antibodies that will bind the sameepitope on NKp46 as antigen binding domains herein can be assessed, e.g.as described in for example, in PCT application numberPCT/EP2016/064537, filed 23 Jun. 2016 (Innate Pharma), the disclosure ofwhich is incorporated herein by reference.

Typically, an anti-NKp46 antibody or NKp46 binding protein providedherein has an affinity for a NKp46 polypeptide (e.g., a NKp46polypeptide as produced in the Examples herein) in the range of about10⁴ to about 10¹¹ M⁻¹ (e.g., about 10⁸ to about 10¹⁰ M⁻¹). For example,in a particular aspect the disclosure provides Anti-NKp46 antibody orNKp46 binding protein that has an average disassociation constant(K_(D)) of less than 1×10⁻⁹ M with respect to NKp46, as determined by,e.g., surface plasmon resonance (SPR) screening (such as by analysiswith a BIAcore™ SPR analytical device). In a more particular exemplaryaspect, the disclosure provides anti-NKp46 antibodies or NKp46 bindingproteins that have a KD of about 1×10⁻⁸ M to about 1×10⁻¹⁰ M, or about1×10⁻⁹ M to about 1×10⁻¹¹ M, for human or cynomolgus NKp46 protein. Inone embodiment, the NKp46 antibodies or NKp46 binding proteins have adisassociation constant (K_(D)) for human and cynomolgus NKp46 proteinthat differs by no more than 1- or 2-log.

Antibodies or NKp46 binding proteins can be characterized for example bya mean KD of no more than about (i.e. better affinity than) 100, 60, 10,5, or 1 nanomolar, preferably sub-nanomolar or optionally no more thanabout 500, 200, 100 or 10 picomolar. KD can be determined for examplefor example by immobilizing recombinantly produced human NKp46 proteinson a chip surface, followed by application of the antibody to be testedin solution.

DNA encoding an antibody or other NKp46 binding proteins that binds anepitope present on NKp46 polypeptides is isolated and placed in anappropriate expression vector for transfection into an appropriate host.The host is then used for the recombinant production of the antibody orother NKp46 binding proteins. For example, DNA can be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of the desired proteins in the recombinant hostcells.

In one embodiment, the proteins and antibodies herein bind the D1 domainof NKp46, the D2 domain of NKp46, or to a region spanning both the D1and D2 domains (at the border of the D1 and D2 domains, the D1/D2junction), of the NKp46 polypeptide of SEQ ID NO: 1. In one embodiment,the proteins or antibodies have an affinity for human NKp46characterized by a K_(D) of less than 10⁻⁸ M, less than 10⁻⁹ M, or lessthan 10⁻¹⁹M.

In another embodiment, the proteins or antibodies bind NKp46 atsubstantially the same epitope on NKp46 as antibody NKp46-1, NKp46-2,NKp46-3 or NKp46-4. In another embodiment, the antibodies at leastpartially overlaps, or includes at least one residue in the segmentbound by NKp46-1, NKp46-2, NKp46-3 or NKp46-4. In one embodiment, allkey residues of the epitope are in a segment corresponding to domain D1or D2. In one embodiment, the antibody binds a residue present in the D1domain as well as a residue present in in the D2 domain. In oneembodiment, the antibodies or proteins bind an epitope comprising 1, 2,3, 4, 5, 6, 7 or more residues in the segment corresponding to domain D1or D2 of the NKp46 polypeptide of SEQ ID NO: 1. In one embodiment, theantibodies or proteins bind domain D1 and bind an epitope comprising 1,2, 3, or 4 of the residues R101, V102, E104 and/or L105. In oneembodiment, the antibodies or proteins bind domain D1/D2 junction andbind an epitope comprising 1, 2, 3, 4 or 5 of the residues K41, E42,E119, Y121 and/or Y194. In one embodiment, the antibodies bind domain D2and bind an epitope comprising 1, 2, 3, or 4 of the residues P132, E133,1135, and/or S136.

The Examples section herein describes the construction of a series ofmutant human NKp46 polypeptides. Binding of anti-NKp46 antibody orproteins to cells transfected with the NKp46 mutants was measured andcompared to the ability of anti-NKp46 antibody or protein to bindwild-type NKp46 polypeptide (SEQ ID NO:1). A reduction in bindingbetween an anti-NKp46 antibody or protein and a mutant NKp46 polypeptideas used herein means that there is a reduction in binding affinity(e.g., as measured by known methods such FACS testing of cellsexpressing a particular mutant, or by Biacore testing of binding tomutant polypeptides) and/or a reduction in the total binding capacity ofthe anti-NKp46 antibody or protein (e.g., as evidenced by a decrease inBmax in a plot of anti-NKp46 antibody concentration versus polypeptideconcentration). A significant reduction in binding indicates that themutated residue is directly involved in binding to the anti-NKp46antibody or protein or is in close proximity to the binding protein whenthe anti-NKp46 antibody or protein is bound to NKp46. An epitope willthus preferably include such residue and may include additional residuesadjacent to such residue.

In some embodiments, a significant reduction in binding means that thebinding affinity and/or capacity between an anti-NKp46 antibody orprotein and a mutant NKp46 polypeptide is reduced by greater than 40%,greater than 50%, greater than 55%, greater than 60%, greater than 65%,greater than 70%, greater than 75%, greater than 80%, greater than 85%,greater than 90% or greater than 95% relative to binding between theantibody or protein and a wild type NKp46 polypeptide (e.g., thepolypeptide shown in SEQ ID NO:1). In certain embodiments, binding isreduced below detectable limits. In some embodiments, a significantreduction in binding is evidenced when binding of an anti-NKp46 antibodyto a mutant NKp46 polypeptide is less than 50% (e.g., less than 45%,40%, 35%, 30%, 25%, 20%, 15% or 10%) of the binding observed between theanti-NKp46 antibody and a wild-type NKp46 polypeptide (e.g., thepolypeptide shown in SEQ ID NO: 1 (or the extracellular domainthereof)). Such binding measurements can be made using a variety ofbinding assays known in the art. A specific example of one such assay isdescribed in the Example section.

In some embodiments, anti-NKp46 antibodies or proteins are provided thatexhibit significantly lower binding for a mutant NKp46 polypeptide inwhich a residue in a wild-type NKp46 polypeptide (e.g., SEQ ID NO:1) issubstituted. In the shorthand notation used here, the format is: Wildtype residue: Position in polypeptide: Mutant residue, with thenumbering of the residues as indicated in SEQ ID NO: 1.

In some embodiments, an anti-NKp46 antibody or protein binds a wild-typeNKp46 polypeptide but has decreased binding to a mutant NKp46polypeptide having a mutation (e.g., an alanine substitution) any one ormore of the residues R101, V102, E104 and/or L105 (with reference to SEQID NO:1) compared to binding to the wild-type NKp46).

In some embodiments, an anti-NKp46 antibody or protein binds a wild-typeNKp46 polypeptide but has decreased binding to a mutant NKp46polypeptide having a mutation (e.g., an alanine substitution) any one ormore of the residues K41, E42, E119, Y121 and/or Y194 (with reference toSEQ ID NO:1) compared to binding to the wild-type NKp46).

In some embodiments, an anti-NKp46 antibody or protein binds a wild-typeNKp46 polypeptide but has decreased binding to a mutant NKp46polypeptide having a mutation (e.g., an alanine substitution) any one ormore of the residues P132, E133, 1135, and/or S136 (with reference toSEQ ID NO:1) compared to binding to the wild-type NKp46).

The amino acid sequence of the heavy and light chain variable region ofparental antibodies NKp46-1, NKp46-2, NKp46-3 and NKp46-4 are listedherein in Table B. Amino acid sequence of the heavy and light chainvariable region of humanized antibodies NKp46-1, NKp46-2, NKp46-3,NKp46-4 and NKp46-9 that displayed high binding affinity for both humanand non-human primate NKp46 are listed herein in Table C.

A heavy chain variable region of a NKp46-1, NKp46-2, NKp46-3, NKp-46-4,NKp46-6 or NKp46-9 antibody may comprise, for the respective antibody: ahuman heavy chain FR1 framework region; a HCDR1 region comprising anamino acid sequence as set forth in Table A, or a sequence of at least4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one ormore of these amino acids may be substituted by a different amino acid;a human heavy chain FR2 framework region; a HCDR2 region comprising anamino acid sequence as set forth in Table A, or a sequence of at least4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one ormore of these amino acids may be substituted by a different amino acid;a human heavy chain FR3 framework region; and a HCDR3 region comprisingan amino acid sequence as set forth in as set forth in Table A, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid. Optionally, the variable region furthercomprises a human heavy chain FR4 framework region.

A light chain variable region of a NKp46-1, NKp46-2, NKp46-3, NKp-46-4,NKp46-6 or NKp46-9 antibody may comprise, for the respective antibody: ahuman light chain FR1 framework region; a LCDR1 region comprising anamino acid sequence as set forth in Table A, or a sequence of at least4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one ormore of these amino acids may be substituted by a different amino acid;a human light chain FR2 framework region; a LCDR2 region comprising anamino acid sequence as set forth in Table A, or a sequence of at least4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one ormore of these amino acids may be substituted by a different amino acid;a human light chain FR3 framework region; and a LCDR3 region comprisingan amino acid sequence as set forth in Table A, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein oneor more of these amino acids may be deleted or substituted by adifferent amino acid. Optionally, the variable region further comprisesa human light chain FR4 framework region.

Examples of VH and VL combinations include:

-   -   (a) a VH comprising a CDR1, 2 and 3 of SEQ ID NO: 3 and a FR1, 2        and 3 of a human IGHV1-69 gene segment, and a VL comprising a        CDR1, 2 and 3 of SEQ ID NO: 4 and a FR1, 2 and 3 of a human        IGKV1-33 gene segment;    -   (b) a VH comprising a CDR1, 2 and 3 of SEQ ID NO: 5 and a FR1, 2        and 3 of a human IGHV4-30-4 gene segment, and a VL comprising a        CDR1, 2 and 3 of SEQ ID NO: 6 and a FR1, 2 and 3 of a human        IGKV1-39 gene segment;    -   (c) a VH comprising a CDR1, 2 and 3 of SEQ ID NO: 7 and a FR1, 2        and 3 of a human IGHV1-69 gene segment, and a VL comprising a        CDR1, 2 and 3 of SEQ ID NO: 8 and a FR1, 2 and 3 of a human        IGKV3-11 and/or IGKV3-15 gene segment;    -   (d) a VH comprising a CDR1, 2 and 3 of SEQ ID NO: 9 and a FR1, 2        and 3 of a human IGHV1-46 and/or a IGHV1-69 gene segment, and a        VL comprising a CDR1, 2 and 3 of SEQ ID NO: 10 and a FR1, 2 and        3 of a human IGKV1-NL1 gene segment; or    -   (e) a VH comprising a CDR1, 2 and 3 of SEQ ID NO: 13 and a FR1,        2 and 3 of a human IGHV4-30-4 gene segment, and a VL comprising        a CDR1, 2 and 3 of SEQ ID NO: 14 and a FR1, 2 and 3 of a human        IGKV1-39 gene segment.

In another aspect, examples of VH and VL combinations include:

-   -   (a) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% identical to the amino acid sequence of the        NKp46-1 H1 or H3 variable domain, and a VL comprising an amino        acid sequence at least 70%, 80%, 90%, 95% or 98% identical to        the amino acid sequence of the NKp46-1 L1 variable domain;    -   (b) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% identical to the amino acid sequence of the        NKp46-2 H1, H2 or H3 variable domain, and a VL comprising an        amino acid sequence at least 70%, 80%, 90%, 95% or 98% identical        to the amino acid sequence of the NKp46-2 L1 variable domain;    -   (c) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% identical to the amino acid sequence of the        NKp46-3 H1, H3 or H4 variable domain, and a VL comprising an        amino acid sequence at least 70%, 80%, 90%, 95% or 98% identical        to the amino acid sequence of the NKp46-3 L1 variable domain;    -   (d) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% identical to the amino acid sequence of the        NKp46-4 H1 variable domain, and a VL comprising an amino acid        sequence at least 70%, 80%, 90%, 95% or 98% identical to the        amino acid sequence of the NKp46-4 L2 variable domain;    -   (e) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% identical to the amino acid sequence of the        NKp46-9 H2 variable domain, and a VL comprising an amino acid        sequence at least 70%, 80%, 90%, 95% or 98% identical to the        amino acid sequence of the NKp46-9 L1 or L2 variable domain; or    -   (f) a VH comprising an amino acid sequence at least 70%, 80%,        90%, 95% or 98% identical to the amino acid sequence of the        NKp46-9 H3 variable domain, and a VL comprising an amino acid        sequence at least 70%, 80%, 90%, 95% or 98% identical to the        amino acid sequence of the NKp46-9 L1 or L2 variable domain.

In one embodiment, the aforementioned CDRs are according to Kabat, e.g.as shown in Table A. In one embodiment, the aforementioned CDRs areaccording to Chotia numbering, e.g. as shown in Table A. In oneembodiment, the aforementioned CDRs are according to IMGT numbering,e.g. as shown in Table A.

In one embodiment, the particular VH or VL comprises an amino acidsubstitution in a human framework (e.g. a back-mutation) at a Kabatposition shown in Example 1; optionally the amino acid residuesubstituted in Example 1 at the corresponding Kabat position can bespecified as being present at the particular position.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO or Table A.

The sequences of the CDRs, according to IMGT, Kabat and Chothiadefinitions systems, have been summarized in Table A below. Thesequences of the variable chains of the antibodies according to theinvention are listed in Table B below. In any embodiment herein, a VL orVH sequence can be specified or numbered so as to contain or lack asignal peptide or any part thereof.

TABLE A HCDR1 HCDR2 HCDR3 CDR SEQ SEQ SEQ mAb definition ID Sequence IDSequence ID Sequence NKp46-1 Kabat 15 DYVIN  18 EIYPGSGTNYYNEKFKA  21RGRYGLYAMDY Chotia 16 GYTFTDY  19 PGSG  22 GRYGLYAMD IMGT 17 GYTFTDYV 20 GYTFTDYVIYPGSGTN  23 ARRGRYGLYAMDY NKp46-2 Kabat 31 SDYAWN  34YITYSGSTSYNPSLES  36 GGYYGSSWGVFAY Chotia 32 GYSITSDY YSG  37GYYGSSWGVFA IMGT 33 GYSITSDYA  35 ITYSGST  38 ARGGYYGSSWGVFAY NKp46-3Kabat 46 EYTMH  49 GISPNIGGTSYNQKFKG  51 RGGSFDY Chotia 47 GYTFTEY PNIG 52 GGSFD IMGT 48 GYTFTEYT  50 ISPNIGGT  53 ARRGGSFDY NKp46-4 Kabat 60SFTMH  63 YINPSSGYTEYNQKFKD  65 GSSRGFDY Chotia 61 GYTFTSF PSSG  66SSRGFD IMGT 62 GYTFTSFT  64 INPSSGYT  67 VRGSSRGFDY NKp46-6 Kabat 73SSWMH  76 HIHPNSGISNYNEKFKG  78 GGRFDD Chotia 74 GYTFTSS PNSG GRFD IMGT75 GYTFTSSW  77 IHPNSGIS  79 ARGGRFDD NKp46-9 Kabat 85 SDYAWN  88YITYSGSTNYNPSLKS  89 CWDYALYAMDC Chotia 86 GYSITSDY YSG  90 WDYALYAMDIMGT 87 GYSITSDYA  35 ITYSGST  91 ARCWDYALYAMDC Bab281 Kabat 97 NYGMN100 WINTNTGEPTYAEEFKG 102 DYLYYFDY Chotia 98 GYTFTNY TNTG 103 YLYYFDIMGT 99 GYTFTNYG 101 INTNTGEP 104 ARDYLYYFDY LCDR1 LCDR2 LCDR3 CDR SEQSEQ SEQ mAb definition ID Sequence ID Sequence ID Sequence NKp46-1 Kabat 24 RASQDISNYLN  27 YTSRLHS  28 QQGNTRPWT Chotia  25 SQDISNY YTS  29YTSGNTRPW IMGT  26 QDISNY YTS  30 YTSQQGNTRPWT NKp46-2 Kabat  39RVSENIYSYLA  42 NAKTLAE  43 QHHYGTPWT Chotia  40 SENIYSY NAK  44 HYGTPWIMGT  41 ENIYSY NAK  45 QHHYGTPWT NKp46-3 Kabat  54 RASQSISDYLH  57YASQSIS  58 QNGHSFPLT Chotia  55 SQSISDY YAS  59 GHSFPL IMGT  56 QSISDYYAS QNGHSFPLT NKp46-4 Kabat  68 RASENIYSNLA  70 AATNLAD  71 QHFWGTPRTChotia SENIYSN AAT  72 FWGTPR IMGT  69 ENIYSN AAT QHFWGTPRT NKp46-6Kabat  80 RASQSISDYLH YASQSIS  82 QNGHSFLMYT Chotia  81 GRFDSQSISDY YAS 83 GHSFLMY IMGT QSISDY YAS  84 YASQNGHSFLMYT NKp46-9 Kabat  92RTSENIYSYLA  93 NAKTLAE  94 QHHYDTPLT Chotia SENIYSY NAK  95 NAKHYDTPLIMGT ENIYSY NAK  96 QHHYDTPLT Bab281 Kabat 105 KASENVVTYVS 108 GASNRYT109 GQGYSYPYT Chotia 106 SENVVTY GAS 110 GYSYPY IMGT 107 ENVVTY GAS 111GQGYSYPYT

TABLE B SEQ ID Antibody NO Amino acid sequence NKp46-1 VH  3QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEIYPGSGTNYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRY GLYAMDYWGQGTSVTVSSNKp46-1 VL  4 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGT KLEIK NKp46-2 VH  5EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTSYNPSLESRISITRDTSTNQFFLQLNSVTTEDTATYYCARGGYY GSSWGVFAYWGQGTLVTVSANKp46-2 VL  6 DIQMTQSPASLSASVGETVTITCRVSENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRESGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPWTFGGGT KLEIK NKp46-3 VH  7EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGISPNIGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGGS FDYWGQGTTLTVSSNKp46-3 VL  8 DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCONGHSFPLTFGAGT KLELK NKp46-4 VH  9QVQLQQSAVELARPGASVKMSCKASGYTFTSFTMHWVKQRPGQGLEWIGYINPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLDSLTSDDSAVYYCVRGSSR GFDYWGQGTLVTVSANKp46-4 VL 10 DIQMIQSPASLSVSVGETVTITCRASENIYSNLAWFQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGIYYCQHEWGTPRTFGGGT KLEIK NKp46-6 VH 11QVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWMHWAKQRPGQGLEWIGHIHPNSGISNYNEKFKGKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARGGRF DDWGAGTTVTVSSNKp46-6 VL 12 DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRESGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFLMYTFGGG TKLEIK NKp46-9 VH 13DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARCWDY ALYAMDCWGQGTSVTVSSNKp46-9 VL 14 DIQMTQSPASLSASVGETVTITCRTSENIYSYLAWCQQKQGKSPQLLVYNAKTLAEGVPSRESGSGSGTHFSLKINSLQPEDFGIYYCQHHYDTPLTFGAGT KLELK

TABLE C VH VL Antibody (SEQ ID NO) (SEQ ID NO) NKp46-1 H1L1 199 201NKp46-1 H3L1 200 201 NKp46-2 H1L1 202 205 NKp46-2 H2L1 203 205 NKp46-2H3L1 204 205 NKp46-3 H1L1 206 209 NKp46-3 H3L1 207 209 NKp46-3 H4L1 208209 NKp46-4 H1L2 210 213 NKp46-4 H2L2 211 213 NKp46-4 H3L2 212 213NKp46-9 H2L1 215 217 NKp46-9 H2L2 215 218 NKp46-9 H3L1 216 217 NKp46-9H3L2 216 218Multi-Specific Antibodies and Polypeptides

Antigen binding domains (ABDs) that bind NKp46 can be derived from theanti-NKp46 CDR, VH and VL sequences provided herein (see section“Anti-NKp46 variable domains”). The antigen binding domains can bearranged one the same or on separate polypeptides, such that they forman antigen binding domain capable of binding NKp46 (e.g. human NKp46 asexpressed at the surface of a cell). The ABDs can be produced, forexample, as an scFv, a tandem scFv, a Bite, a DART, a Fab, a F(ab)₂, anantibody, a bispecific antibody, or a monomeric or multimeric Fcprotein.

For the construction of multi-specific proteins (e.g. antibodies, tandemscFv, BiTe, DART, Fc proteins), antigen binding domains that bind anantigen of interest (other than NKp46) can be readily derived a varietyof immunoglobulin or non-immunoglobulin scaffolds, for exampleaffibodies based on the Z-domain of staphylococcal protein A, engineeredKunitz domains, monobodies or adnectins based on the 10th extracellulardomain of human fibronectin Ill, anticalins derived from lipocalins,DARPins (designed ankyrin repeat domains, multimerized LDLR-A module,avimers or cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra(2009) Current Opinion in Chemical Biology 13:245-255, the disclosure ofwhich is incorporated herein by reference.

Variable domains that bind an antigen of interest (other than NKp46) canbe derived from an antibody, for example in the form of associated VLand VH domains found on two polypeptide chains, or single chain antigenbinding domains such as scFv, a V_(H) domain, a V_(L) domain, a dAb, aV-NAR domain or a V_(H)H domain. The an antigen binding domain (e.g,ABD₁ and ABD₂) can also be readily derived from antibodies as a Fab.

Typically, antibodies are initially obtained by immunization of anon-human animal, e.g., a mouse, with an immunogen comprising apolypeptide, or a fragment or derivative thereof, typically animmunogenic fragment, for which it is desired to obtain antibodies (e.g.a human polypeptide). The step of immunizing a non-human mammal with anantigen may be carried out in any manner well known in the art forstimulating the production of antibodies in a mouse (see, for example,E. Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1988), the entiredisclosure of which is herein incorporated by reference).

Antigen binding domains (ABDs) that bind antigens of interest (otherthan NKp46) for use in a multispecific polypeptide can be selected basedon the desired cellular target, and may include for example cancerantigens, bacterial or viral antigens, etc. As used herein, the term“bacterial antigen” includes, but is not limited to, intact, attenuatedor killed bacteria, any structural or functional bacterial protein orcarbohydrate, or any peptide portion of a bacterial protein ofsufficient length (typically about 8 amino acids or longer) to beantigenic. Examples include gram-positive bacterial antigens andgram-negative bacterial antigens. In some embodiments the bacterialantigen is derived from a bacterium selected from the group consistingof Helicobacter species, in particular Helicobacter pyloris; Boreliaspecies, in particular Borelia burgdorferi; Legionella species, inparticular Legionella pneumophilia; Mycobacteria s species, inparticular M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M.gordonae; Staphylococcus species, in particular Staphylococcus aureus;Neisseria species, in particular N. gonorrhoeae, N. meningitidis;Listeria species, in particular Listeria monocytogenes; Streptococcusspecies, in particular S. pyogenes, S. agalactiae; S. faecalis; S.bovis, S. pneumonias; anaerobic Streptococcus species; pathogenicCampylobacter species; Enterococcus species; Haemophilus species, inparticular Haemophilus influenzue; Bacillus species, in particularBacillus anthracis; Corynebacterium species, in particularCorynebacterium diphtheriae; Erysipelothrix species, in particularErysipelothrix rhusiopathiae; Clostridium species, in particular C.perfringens, C. tetani; Enterobacter species, in particular Enterobacteraerogenes, Klebsiella species, in particular Klebsiella 1S pneumoniae,Pasteurella species, in particular Pasteurella multocida, Bacteroidesspecies; Fusobacterium species, in particular Fusobacterium nucleatum;Streptobacillus species, in particular Streptobacillus moniliformis;Treponema species, in particular Treponema pertenue; Leptospira;pathogenic Escherichia species; and Actinomyces species, in particularActinomyces israelli.

As used herein, the term “viral antigen” includes, but is not limitedto, intact, attenuated or killed whole virus, any structural orfunctional viral protein, or any peptide portion of a viral protein ofsufficient length (typically about 8 amino acids or longer) to beantigenic. Sources of a viral antigen include, but are not limited toviruses from the families: Retroviridae (e.g., human immunodeficiencyviruses, such as HIV-1 (also referred to as HTLV-III, LAV orHTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses);Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g., African swine fever virus); and unclassified viruses(e.g., the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), Hepatitis C; Norwalk and related viruses, andastroviruses). Alternatively, a viral antigen may be producedrecombinantly.

As used herein, the terms “cancer antigen” and “tumor antigen” are usedinterchangeably and refer to antigens (other than NKp46) that aredifferentially expressed by cancer cells or are expressed by non-tumoralcells (e.g. immune cells) having a pro-tumoral effect (e.g. animmunosuppressive effect), and can thereby be exploited in order totarget cancer cells. Cancer antigens are antigens which can potentiallystimulate apparently tumor-specific immune responses. Some of theseantigens are encoded, although not necessarily expressed, or expressedat lower levels or less frequently, by normal cells. These antigens canbe characterized as those which are normally silent (i.e., notexpressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses. Still other cancer antigens can be expressed onimmune cells capable of mediating a pro-tumoral effect, e.g. a monocyteor a macrophage, optionally a suppressor T cell, regulatory T cell, ormyeloid-derived suppressor cell.

The cancer antigens are usually normal cell surface antigens which areeither over-expressed or expressed at abnormal times, or are expressedby a targeted population of cells. Ideally the target antigen isexpressed only on proliferative cells (e.g., tumor cells) or pro-tumoralcells (e.g. immune cells having an immunosuppressive effect), howeverthis is rarely observed in practice. As a result, target antigens are inmany cases selected on the basis of differential expression betweenproliferative/disease tissue and healthy tissue. Example of cancerantigens include: Receptor Tyrosine Kinase-like Orphan Receptor 1(ROR1), Cripto, CD4, CD20, CD30, CD19, CD38, CD47, Glycoprotein NMB,CanAg, Her2 (ErbB2/Neu), a Siglec family member, for example CD22(Siglec2) or CD33 (Siglec3), CD79, CD138, CD171, PSCA, L1-CAM, PSMA(prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70,E-selectin, EphB2, Melanotransferin, Mud 6 and TMEFF2. Examples ofcancer antigens also include Immunoglobulin immunoglobulin superfamily(IgSF) such as cytokine receptors, Killer-Ig Like Receptor, CD28 familyproteins, for example, Killer-Ig Like Receptor 3DL2 (KIR3DL2), B7-H3,B7-H4, B7-H6, PD-L1, IL-6 receptor. Examples also include MAGE,MART-1/Melan-A, gp100, major histocompatibility complex class I-relatedchain A and B polypeptides (MICA and MICB), adenosine deaminase-bindingprotein (ADAbp), cyclophilin b, colorectal associated antigen(CRC)-0017-1A/GA733, protein tyrosine kinase 7 (PTK7), receptor proteintyrosine kinase 3 (TYRO-3), nectins (e.g. nectin-4), majorhistocompatibility complex class I-related chain A and B polypeptides(MICA and MICB), proteins of the UL16-binding protein (ULBP) family,proteins of the retinoic acid early transcript-1 (RAET1) family,carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 andCAP-2, etv6, am11, prostate specific antigen (PSA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens, GAGE-family oftumor antigens, anti-Mullerian hormone Type II receptor, delta-likeligand 4 (DLL4), DRS, ROR1 (also known as Receptor Tyrosine Kinase-LikeOrphan Receptor 1 or NTRKR1 (EC 2.7.10.1), BAGE, RAGE, LAGE-1, NAG,GnT-V, MUM-1, CDK4, MUC family, VEGF, VEGF receptors, Angiopoietin-2,PDGF, TGF-alpha, EGF, EGF receptor, members of the human EGF-likereceptor family, e.g., HER-2/neu, HER-3, HER-4 or a heterodimericreceptor comprised of at least one HER subunit, gastrin releasingpeptide receptor antigen, Muc-1, CA125, integrin receptors, αvβ3integrins, α5β1 integrins, αIIbβ3-integrins, PDGF beta receptor,SVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, CSF1R (tumor-associatedmonocytes and macrophages), α-fetoprotein, E-cadherin, α-catenin,β-catenin and γ-catenin, p120ctn, PRAME, NY-ESO-1, cdc27, adenomatouspolyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15,gp75, GM2 and GD2 gangliosides, viral products such as humanpapillomavirus proteins, imp-1, P1A, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, although this is notintended to be exhaustive. In one aspect, the antigen of interest is anantigen (e.g. any one of the antigens listed above) capable ofundergoing intracellular internalization, for example when bound by anconventional human IgG1 antibody, either in the presence of absence ofFcγ receptor cells. In one aspect, the antigen of interest is a CD19polypeptide; in one aspect, the multispecific protein comprises an scFvthat binds CD19 comprising an amino acid sequence which is at least 60%,70%, 80%, 85%, 90% or 95% identical to the sequence of the anti-CD19scFv of the Examples herein, or that comprises the heavy and light chainCDR1, -2 and -3 of the anti-CD19 heavy and light chain variable regionsshown herein.

In one embodiment, the ABD that binds an antigen of interest is derivedfrom (e.g. comprises the hypervariable region of, or comprises the CDRsof) a parental antibody that binds an antigen of interest (e.g. a murineantibody, a human antibody) which, when bound to its antigenic target(the antigen of interest on cells), increases or induces down-modulationor intracellular internalization of the antigen of interest. In oneembodiment, the antigen of interest is a cancer antigen, e.g. one of thecancer antigens listed above known to internalize (e.g. Immunoglobulinimmunoglobulin superfamily (IgSF) members, for example cytokine receptoralpha or beta chains, Killer-Ig Like Receptors, CD28 family proteins,B7-H3, B7-H4, B7-H6, KIR3DL2, PTK7, ROR1, L1-CAM, Siglec family members,EGF receptor and EGF-like receptor family members, EGFR, HER-2,integrins, anti-Mullerian hormone Type II receptor, CSF-1R, and others)In one embodiment, the antigen target is a polypeptide present on animmune cell capable of mediating a pro-tumoral effect, e.g. a monocyteor a macrophage, optionally a suppressor T cell, regulatory T cell, ormyeloid-derived suppressor cell.

In one embodiment, the non-NKp46 ABD binds to a cancer antigen, a viralantigen, a microbial antigen, or an antigen present on an infected cell(e.g. virally infected) or on a pro-inflammatory immune cell.

Once appropriate antigen binding domains having desired specificityand/or activity are identified, DNA encoding each of the or ABD can beseparately placed, in suitable arrangements, in an appropriateexpression vector, together with DNA encoding any elements such as anenzymatic recognition tag, CH1, Cκ, CH2 and/or CH3 domains and any otheroptional elements (e.g. DNA encoding a hinge region) for transfectioninto an appropriate host. ABDs will be arranged in an expression vector,or in separate vectors as a function of which type of polypeptide is tobe produced, so as to produce the Fc-polypeptides having the desireddomains operably linked to one another. The host is then used for therecombinant production of the multispecific polypeptide.

For example, a polypeptide fusion product can be produced from a vectorin which the first of the two ABD is operably linked (e.g. directly, viaa heavy or light chain CH1, CK or Cλ constant region and/or hingeregion) to the N-terminus of a CH2 domain, and the CH2 domain isoperably linked at its C-terminus to the N-terminus a CH3 domain. Thesecond of the two ABD can be linked to the polypeptide at eitherterminus, or can be on a second polypeptide chain that forms a dimer,e.g. heterodimer, with the polypeptide comprising the first ABD. Thepolypeptide may comprise a full length Fc domain.

The multispecific polypeptide can then be produced in an appropriatehost cell or by any suitable synthetic process. A host cell chosen forexpression of the multispecific polypeptide is an important contributorto the final composition, including, without limitation, the variationin composition of the oligosaccharide moieties decorating the protein inthe immunoglobulin CH2 domain. The host cell may, for example, be ofmammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21,CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma, yeast,insect or plant cells, or any derivative, immortalized or transformedcell thereof.

A range of different protein formats can be prepared using theNKp46-binding VH-VL pairs of the disclosure, including monomeric,heterodimeric, hetertrimeric and tetrameric multispecific proteins.Included, without limitation, are any of a variety of formats describedin for example, in PCT application number PCT/EP2016/064537, filed 23Jun. 2016 (Innate Pharma), the disclosure of which is incorporatedherein by reference.

Monomeric Proteins

In one example, a multispecific protein comprises, in a singlepolypeptide chain, a first antigen binding domain that binds to NKp46(e.g. an ABD comprising a VH and a VL disclosed herein, separated by a(poly)peptide linker) and a second antigen binding domain that binds anantigen other than NKp46. In one embodiment, the protein or polypeptideis or comprises an scFv comprising the anti-NKp46 VH and VL of thedisclosure, or a tandem scFv comprising the scFv that binds NKp46 and asecond scFv that binds NKp46 or an antigen of interest other than NKp46,linked by a (poly)peptide linker optionally fused to another polypeptideor amino acid sequence.

Examples of such single chain antigen binding proteins include BiTE andDART protein formats. An scFv-based bispecific antibody construct, knownas BITE® (Bispecific T-cell Engager) employs a single polypeptidecontaining two antigen-binding domains, each contributed by a pair of VHand VL, linked in tandem via a flexible linker (see, e.g., Nagorsen etal., 2009, Leukemia & Lymphoma 50:886-91; Amann et al., 2009, JImmunother 32:453-64; Baeuerle and Reinhardt, 2009, Cancer Res69:4941-44). Another bispecific antibody called DART® (Dual-AffinityRe-Targeting) utilizes a disulfide-stabilized diabody design (see, e.g.,Moore et al., 2011, Blood 117:4542-51; Veri et al., 2010, ArthritisRheum 62:1933-43).

In one embodiment, the single polypeptide chain further comprises an Fcdomain (e.g. a full length Fc domain or a portion thereof), optionallywherein the Fc domain is interposed between the first and second antigenbinding domains.

In one aspect of any embodiment, the first antigen binding domain and/orthe second antigen binding domain comprise a scFv, optionally where thescFv comprises human framework amino acid sequences. In one embodiment,provided is a monomeric bispecific Fc-derived polypeptide comprising:(a) a first scFv that binds to NKp46; (b) a second scFv that binds anantigen other than NKp46; and, optionally, (c) at least a portion of ahuman Fc domain. Optionally the Fc domain (i) does not dimerize withanother Fc-derived polypeptide and (ii) is capable of binding to humanFcRn. Optionally, the Fc domain is interposed between the first andsecond scFv.

When the polypeptide fusion product comprising the two ABDs and at leasta portion of an Fc domain is a monomer, the CH3 domains may be arrangedand/or comprise amino acid modification to prevent CH3-CH3 dimerization.In one embodiment, the CH3 domain comprises mutations in the dimerinterface to prevent interchain CH3-CH3 dimerization. In anotherembodiment, the CH3 domain is a tandem CH3 domain (or the Fc domaincomprises a tandem CH3 domain) to prevent interchain CH3-CH3dimerization. Such monomers will retain partial FcRn binding (compared,e.g., to a wild type full length human IgG1 antibody). Optionally themonomeric polypeptide is capable of binding to human FcRn withintermediate affinity, e.g. retains binding to FcRn but has decreasedbinding to a human FcRn receptor compared to a full-length wild typehuman IgG1 antibody. The Fc moiety may further comprise one or moreamino acid modifications, e.g. in the CH2 domain, that further decreasesor substantially abolishes binding to one or more Fcγ receptors.

In one configuration, the monomeric Fc-derived polypeptides that have atleast a portion of a human Fc domain can advantageously comprise a CH2domain and a CH3 domain, wherein said CH3 domain comprises a modifiedCH3 dimer interface (e.g. a mutations in the CH3 dimer interface) toprevent dimerization with another Fc-derived polypeptide. See e.g.format 1 and 2 in FIG. 2A). In one embodiment of any of the polypeptidesor methods herein, the CH3 domain comprises an amino acid substitutionat 1, 2, 3, 4, 5, 6 or 7 of the positions L351, T366, L368, P395, F405,T407 (or Y407) and/or K409 (EU numbering as in Kabat).

Another configuration for a CH3 domain that can be used in a monomericmultispecific protein is a tandem CH3 domain (see e.g. format 3 and 4 inFIG. 2A). A tandem CH3 domain comprises a first and a second CH3 domain,wherein the two CH3 domains associate with one another via non-covalentinteractions. In one embodiment, the two CH3 domains associate with oneanother via the CH3 dimerization interface of each CH3 domain. In oneembodiment, the polypeptide chain does not dimerize with anotherpolypeptide chain comprising an Fc domain. An Fc domain that comprise atandem CH3 domain will interact with neonatal Fc receptor (FcRn) butwill have low or no binding to human FCγ receptors, notably CD16.

Multimeric Proteins

Multimeric bispecific proteins such as heterodimers, heterotrimers andtetramers (the latter including for example antibodies with two heavychains and two light chains) that comprise an ABD that binds NKp46 (e.g.an ABD comprising a VH and a VL disclosed herein) can be producedaccording to a variety of formats.

In one embodiment, a multimeric protein or polypeptide is a tetramericantibody made up of two heavy chains comprising variable regions (or 1,2 or 3 CDRs thereof) derived from two different parental antibodies, andtwo light chains comprising variable regions (or 1, 2 or 3 CDRs thereof)derived from two different parental antibodies. Such a tetramer maycomprise (a) two heavy chains each comprising a variable region, a CH1domain, hinge and an Fc domain, and (b) two antibody light chains eachcomprising a light chain variable region and a CK domain, wherein oneheavy chain variable region together with a light chain variable regionbinds to NKp46 and the other heavy chain variable region together with alight chain variable region bind an antigen of interest.

One advantageous way of making multimeric proteins is through theassembly of different polypeptide chains that each comprise at least oneheavy or light chain variable domain fused to a human CH1 or Cκ constantdomain (a V—(CH1/Cκ) unit), wherein the protein chains undergo CH1-Cκdimerization and are bound to one another by non-covalent bonds andoptionally further by disulfide bonds formed between respective CH1 andCκ domain. In one embodiment, provided is an isolated or purifiedheterodimeric or heterotimeric protein that binds a first and secondantigen, wherein the protein comprises at least two or three polypeptidechains each comprising a V—(CH1/Cκ) unit, whereby the chains are boundto one another by non-covalent bonds and optionally further by disulfidebonds between CH1 and Cκ domains, optionally, whereby the chains arefurther bound by non-covalent bonds between respective variable regionsand CH3 domains of the Fc portion.

The variable and constant regions can be selected and configured suchthat each chain will preferentially associate with its desiredcomplementary partner chain. The resulting multimeric protein willtherefore be simple to produce using conventional production methodsusing recombinant host cells. The choice of which VH, VL to associatewith a CH1 and Cκ in a unit is based on affinity between the units to bepaired so as to drive the formation of the desired multimer. Theresulting multimer will be bound by non-covalent bonds betweencomplementary VH and VL domains, by non-covalent bonds betweencomplementary CH1 and Cκ domains, and optionally by disulfide bondingbetween complementary CH1 and Cκ domains and/or optionally further bydisulfide bonds between complementary hinge domains). VH-VL associationsare stronger than VH-VH or VL-VL, consequently, as shown herein, one canplace a VH or a VL next to either a CH1 or a Cκ, and the resulting V—Cunit will partner preferably with its V—C counterpart. For example VH-Cκwill pair with VL-CH1 preferentially over VH-CH1. Additionally, byincluding an Fc domain, preferred chain pairing is further improved, asthe two Fc-containing chains will be bound by non-covalent bonds betweenCH3 domains of the Fc domains. The different V—C combinations,optionally further combined with Fc pairing thereby provides tools tomake heteromultimeric proteins.

In one embodiment, a heteromultimeric polypeptide or protein comprises amonomeric Fc domain (e.g. the second polypeptide does not comprise an Fcdomain), optionally wherein the Fc domain comprises a CH3 domain with anamino acid mutation to prevent CH3-CH3 dimerization or a tandem CH3domain.

In another embodiment, the above heteromultimeric polypeptide or proteincomprises a dimeric Fc domain capable of binding to human CD16, e.g. ahuman Fc domain comprising N-linked glycosylation at amino acid residueN297 (Kabat EU numbering).

A heterodimer can for example have the configuration as follows (seealso Examples of such proteins shown as formats 2, 11 and 12 shown inFIGS. 2A and 2C):

wherein one of V_(a-1) of the first polypeptide chain and V_(b-1) of thesecond polypeptide chain is a light chain variable domain and the otheris a heavy chain variable domain, and wherein one of V_(a-2) and V_(b-2)is a light chain variable domain and the other is a heavy chain variabledomain.

The heterodimer can in another example have the configuration as follows(see also Examples of such proteins shown as format 10 shown in FIG.2B):

wherein one of V_(a-1) of the first polypeptide chain and V_(b-1) of thesecond polypeptide chain is a light chain variable domain and the otheris a heavy chain variable domain, and wherein one of V_(a-2) and V_(b-2)is a light chain variable domain and the other is a heavy chain variabledomain.

The resulting heterodimer can in another example have the configurationas follows (see also Examples of such proteins shown as formats 13 and14 shown in FIGS. 2D and 2E):

wherein one of V_(a-1) of the first polypeptide chain and V_(b-1) of thesecond polypeptide chain is a light chain variable domain and the otheris a heavy chain variable domain, and wherein one of V_(a-2) and V_(b-2)is a light chain variable domain and the other is a heavy chain variabledomain.

Heterotrimeric proteins can for example be formed by using a central(first) polypeptide chain comprising a first variable domain (V) fusedto a first CH1 or CK constant region, a second variable domain (V) fusedto a second CH1 or CK constant region, and an Fc domain or portionthereof interposed between the first and second variable domains (i.e.the Fc domain is interposed between the first and second (V—(CH1/CK)units. For example, a central polypeptide chain for use in aheterotrimeric protein can have the domain arrangements (N- toC-terminal) as follows:V_(a-1)—(CH1 or CK)_(a)-Fc domain-V_(a-2)—(CH1 or CK)_(b).

A second polypeptide chain can then comprise a domain arrangement (N- toC-terminal):V_(b-1)—(CH1 or CK)_(c),orV_(b-1)—(CH1 or CK)_(c)-Fc domain

such that the (CH1 or CK)_(c) dimerizes with the (CH1 or CK)_(a) on thecentral chain, and the V_(a-1) and V_(b-1) form an antigen bindingdomain.

A third polypeptide chain can then comprise a domain arrangement (N- toC-terminal):V_(b-2)—(CH1 or CK)_(d),

such that the (CH1 or CK)_(d) dimerizes with the (CH1 or CK)_(b) unit onthe central chain, and the V_(a-2) and V_(b-2) form an antigen bindingdomain.

An example of a configuration of a resulting heterotrimer with a dimericFc domain (also shown as formats 5, 6, 7 and 16 in FIGS. 2D and 2E) hasa domain arrangement:

An example of a configuration of a resulting heterotrimer with amonomeric Fc domain (also shown as formats 8, 9 and 17 in FIGS. 2B and2C) has a domain arrangement:

Thus, in a configuration of a trimer polypeptide, the first polypeptidecan have two variable domains that each form an antigen binding domainwith a variable domain on a separate polypeptide chain (i.e. thevariable domain of the second and third chains), the second polypeptidechain has one variable domain, and the third polypeptide has onevariable domain.

A trimeric polypeptide may comprise:

-   -   (a) a first polypeptide chain comprising a first variable        domain (V) fused to a first CH1 of CK constant region, a second        variable domain (V) fused to a second CH1 of CK constant region,        and an Fc domain or portion thereof interposed between the first        and second variable domains;    -   (b) a second polypeptide chain comprising a variable domain        fused at its C-terminus to a CH1 or CK constant region selected        to be complementary to the first CH1 or CK constant region of        the first polypeptide chain such that the first and second        polypeptides form a CH1-CK heterodimer, and optionally an Fc        domain; and    -   (c) a third polypeptide chain comprising a variable domain fused        (e.g. at its C-terminus) to a CH1 or CK constant region, wherein        the variable domain and the constant region are selected to be        complementary to the second variable domain and second CH1 or CK        constant region of the first polypeptide chain such that the        first and third polypeptides form a CH1-CK heterodimer bound by        non-covalent bonds and optionally further by disulfide bond(s)        formed between the CH1 or CK constant region of the third        polypeptide and the second CH1 or CK constant region of the        first polypeptide, but not between the CH1 or CK constant region        of the third polypeptide and the first CH1 or CK constant region        of the first polypeptide;

wherein the first, second and third polypeptides form a CH1-CKheterotrimer, and wherein the first variable domain of the firstpolypeptide chain and the variable domain of the second polypeptidechain form an antigen binding domain specific for a first antigen ofinterest, and the second variable domain of the first polypeptide chainand the variable domain on the third polypeptide chain form an antigenbinding domain specific for a second antigen of interest. One of the twoantigens of interest will be NKp46, and the ABD that binds NKp46comprises a VH-VL variable domain pair of the disclosure.

Examples of domain arrangement for the trimeric bispecific polypeptideformed from include but are not limited to:

(second polypeptide) (first polypeptide) (third polypeptide)

(second polypeptide) (first polypeptide) (third polypeptide)

(second polypeptide) (first polypeptide) (third polypeptide)

(second polypeptide) (first polypeptide) (third polypeptide)

(second polypeptide) (first polypeptide) (third polypeptide)

(second polypeptide) (first polypeptide) (third polypeptide)

In any of the domain arrangements, the Fc domain may comprise a CH2-CH3unit (a full length CH2 and CH3 domain or a fragment thereof). Inheterodimers or heterotrimers comprising two chains with Fc domains (adimeric Fc domain), the CH3 domain will be capable of CH3-CH3dimerization (e.g. a wild-type CH3 domain). In heterodimers orheterotrimers comprising only one chain with an Fc domain (monomeric Fcdomain), the Fc domain will be incapable of CH3-CH3 dimerization; forexample the CH3 domain(s) will have amino acid modification(s) in theCH3 dimer interface or the Fc domain will comprise a tandem CH3 domainincapable of CH3-CH3 dimerization.

In some exemplary configurations, the multispecific protein can betetramers, e.g. heterodimeric tetramers with two light chains and twodifferent heavy chains, wherein the heavy chains are engineered forheterodimerization. Such proteins can be prepared as described, forexample, in PCT application number PCT/EP2016/064537, filed 23 Jun. 2016(Innate Pharma), the disclosure of which is incorporated herein byreference.

In any protein of the disclosure, a hinge region will typically bepresent on a polypeptide chain between a CH1 domain and a CH2 domain,and/or can be present between a CK domain and a CH2 domain. A hingeregion can optionally be replaced for example by a suitable linkerpeptide.

The proteins domains described in the present disclosure can optionallybe specified as being from N- to C-terminal. Protein arrangements of thedisclosure for purposes of illustration are shown from N-terminus (onthe left) to C-terminus. Domains can be referred to as fused to oneanother (e.g. a domain can be said to be fused to the C-terminus of thedomain on its left, and/or a domain can be said to be fused to theN-terminus of the domain on its right).

The proteins domains described in the present disclosure can be fused toone another directly or via intervening amino acid sequences. Forexample, a CH1 or CK domain will be fused to an Fc domain (or CH2 or CH3domain thereof) via a linker peptide, optionally a hinge region or afragment thereof. In another example, a VH or VK domain will be fused toa CH3 domain via a linker peptide. VH and VL domains linked to anotherin tandem will be fused via a linker peptide (e.g. as an scFv). VH andVL domains linked to an Fc domain will be fused via a linker peptide.Two polypeptide chains will be bound to one another (indicated by “|”)by non-covalent bonds and optionally further by interchain disulfidebonds formed between cysteine residues within complementary CH1 and CKdomains.

Linkers and Fc domains are described in more detail, for example, in PCTapplication number PCT/EP2016/064537, filed 23 Jun. 2016 (InnatePharma), the disclosure of which is incorporated herein by reference.

Once the multispecific protein is produced it can be assessed forbiological activity.

In one aspect of any embodiment herein, a multispecific protein iscapable of inducing activation of an NKp46-expressing cell (e.g. an NKcell, a reporter cell) when the protein is incubated in the presence ofthe NKp46-expressing cell (e.g. purified NK cells) and a target cellthat expresses the antigen of interest).

In one aspect of any embodiment herein, a multispecific protein iscapable of inducing NKp46 signaling in an NKp46-expressing cell (e.g. anNK cell, a reporter cell) when the protein is incubated in the presenceof the NKp46-expressing cell (e.g. purified NK cells) and a target cellthat expresses the antigen of interest).

Optionally, NK cell activation or signaling in characterized byincreased expression of a cell surface marker of activation, e.g. CD107,CD69, etc.

Activity can be measured for example by bringing target cells andNKp46-expressing cells into contact with one another, in presence of themultispecific polypeptide. In one example, aggregation of target cellsand NK cells is measured. In another example, the multispecific proteinmay, for example, be assessed for the ability to cause a measurableincrease in any property or activity known in the art as associated withNK cell activity, respectively, such as marker of cytotoxicity (CD107)or cytokine production (for example IFN-γ or TNF-α), increases inintracellular free calcium levels, the ability to lyse target cells in aredirected killing assay, etc. Assays for activity are further describedin more detail, for example, in PCT application numberPCT/EP2016/064537, filed 23 Jun. 2016 (Innate Pharma), the disclosure ofwhich is incorporated herein by reference.

Uses of Compounds

Compounds according to the disclosure that comprise an antigen bindingdomain that binds NKp46 can be used in a variety of applications,including, e.g. to bind, detect, eliminate, purify or modulate theactivity of NKp46 polypeptides and/or cells that express NKp46polypeptide (e.g. NK cells).

In one aspect, provided are the use of any of the compounds definedherein for the manufacture of a pharmaceutical preparation for thetreatment or diagnosis of a mammal in need thereof. Provided also arethe use any of the compounds defined above as a medicament or an activecomponent or active substance in a medicament. In a further aspectprovided is a method for preparing a pharmaceutical compositioncontaining a compound as defined above, to provide a solid or a liquidformulation for administration orally, topically, or by injection. Sucha method or process at least comprises the step of mixing the compoundwith a pharmaceutically acceptable carrier.

In one aspect, provided is a method to treat, prevent or more generallyaffect a predefined condition by exerting a certain effect, or detect acertain condition using a multispecific protein described herein, or a(pharmaceutical) composition comprising such.

For example, in one aspect, the invention provides a method of restoringor potentiating the activity of NKp46⁺ NK cells in a patient in needthereof (e.g. a patient having a cancer, or a viral or bacterialinfection), comprising the step of administering a multispecific proteindescribed herein to said patient. In one embodiment, the method isdirected at increasing the activity of NKp46+ lymphocytes in patientshaving a disease in which increased lymphocyte (e.g. NK cell) activityis beneficial or which is caused or characterized by insufficient NKcell activity, such as a cancer, or a viral or microbial/bacterialinfection.

The polypeptides described herein can be used to prevent or treatdisorders that can be treated with antibodies, such as cancers, solidand non-solid tumors, hematological malignancies, infections such asviral infections, and inflammatory or autoimmune disorders.

In one embodiment, the antigen of interest (the non-NKp46 antigen) is anantigen expressed on the surface of a malignant cell of a type of cancerselected from the group consisting of: carcinoma, including that of thebladder, head and neck, breast, colon, kidney, liver, lung, ovary,prostate, pancreas, stomach, cervix, thyroid and skin, includingsquamous cell carcinoma; hematopoietic tumors of lymphoid lineage,including leukemia, acute lymphocytic leukemia, acute lymphoblasticleukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma,non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma;hematopoietic tumors of myeloid lineage, including acute and chronicmyelogenous leukemias and promyelocytic leukemia; tumors of mesenchymalorigin, including fibrosarcoma and rhabdomyoscarcoma; other tumors,including neuroblastoma and glioma; tumors of the central and peripheralnervous system, including astrocytoma, neuroblastoma, glioma, andschwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscaroma, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma, hematopoietic tumors of lymphoidlineage, for example T-cell and B-cell tumors, including but not limitedto T-cell disorders such as T-prolymphocytic leukemia (T-PLL), includingof the small cell and cerebriform cell type; large granular lymphocyteleukemia (LGL) preferably of the T-cell type; Sezary syndrome (SS);Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal)T-cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinalT-cell lymphoma; T-lymphoblastic; and lymphoma/leukaemia (T-Lbly/T-ALL).

In one embodiment, polypeptides described herein can be used to preventor treat a cancer selected from the group consisting of: carcinoma,including that of the bladder, head and neck, breast, colon, kidney,liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid andskin, including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including neuroblastoma and glioma; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscaroma, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma. Other exemplary disorders thatcan be treated according to the invention include hematopoietic tumorsof lymphoid lineage, for example T-cell and B-cell tumors, including butnot limited to T-cell disorders such as T-prolymphocytic leukemia(T-PLL), including of the small cell and cerebriform cell type; largegranular lymphocyte leukemia (LGL) preferably of the T-cell type; Sezarysyndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHLhepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma(pleomorphic and immunoblastic subtypes); angio immunoblastic T-celllymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic (Ki 1+) largecell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL).

In one example, the tumor antigen is an antigen expressed on the surfaceof a lymphoma cell or a leukemia cell, and the multispecific protein isadministered to, and/or used for the treatment of, an individual havinga lymphoma or a leukemia. Optionally, the tumor antigen is selected fromCD19, CD20, CD22, CD30 or CD33.

In one aspect, the methods of treatment comprise administering to anindividual a multispecific protein described herein in a therapeuticallyeffective amount, e.g., for the treatment of a disease as disclosedherein, for example a cancer selected from the group above. Atherapeutically effective amount may be any amount that has atherapeutic effect in a patient having a disease or disorder (orpromotes, enhances, and/or induces such an effect in at least asubstantial proportion of patients with the disease or disorder andsubstantially similar characteristics as the patient).

In one embodiment, the multispecific protein described herein may beused as monotherapy (without other therapeutic agents), or in combinedtreatments with one or more other therapeutic agents, including agentsnormally utilized for the particular therapeutic purpose for which theantibody is being administered. The additional therapeutic agent willnormally be administered in amounts and treatment regimens typicallyused for that agent in a monotherapy for the particular disease orcondition being treated. Such therapeutic agents when used in thetreatment of cancer, include, but are not limited to anti-cancer agentsand chemotherapeutic agents; in the treatment of infectious disease,include, but are not limited to anti-viral agents and anti-biotics.

The proteins and/or polypeptides disclosed herein can be included inkits. The kits may optionally further contain any number of polypeptidesand/or other compounds, e.g., 1, 2, 3, 4, or any other number ofproteins and/or polypeptides and/or other compounds. It will beappreciated that this description of the contents of the kits is notlimiting in any way. For example, the kit may contain other types oftherapeutic compounds. Optionally, the kits also include instructionsfor using the proteins and/or polypeptides, e.g., detailing theherein-described methods.

Also provided are pharmaceutical compositions comprising the compoundsas defined above. A compound may be administered in purified formtogether with a pharmaceutical carrier as a pharmaceutical composition.The form depends on the intended mode of administration and therapeuticor diagnostic application. The pharmaceutical carrier can be anycompatible, nontoxic substance suitable to deliver the compounds to thepatient. Pharmaceutically acceptable carriers are well known in the artand include, for example, aqueous solutions such as (sterile) water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters,alcohol, fats, waxes, and inert solids A pharmaceutically acceptablecarrier may further contain physiologically acceptable compounds thatact for example to stabilize or to increase the absorption of thecompounds Such physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the compositionPharmaceutically acceptable adjuvants, buffering agents, dispersingagents, and the like, may also be incorporated into the pharmaceuticalcompositions.

The compounds can be administered parenterally. Preparations of thecompounds for parenteral administration must be sterile. Sterilizationis readily accomplished by filtration through sterile filtrationmembranes, optionally prior to or following lyophilization andreconstitution. The parenteral route for administration of compounds isin accord with known methods, e.g. injection or infusion by intravenous,intraperitoneal, intramuscular, intraarterial, or intralesional routes.The compounds may be administered continuously by infusion or by bolusinjection. A typical composition for intravenous infusion could be madeup to contain 100 to 500 ml of sterile 0.9% NaCl or 5% glucoseoptionally supplemented with a 20% albumin solution and 1 mg to 10 g ofthe compound, depending on the particular type of compound and itsrequired dosing regimen. Methods for preparing parenterallyadministrable compositions are well known in the art.

EXAMPLES Example 1 Generation of Anti-huNKp46 Antibodies

Part A: Generation of Anti-huNKp46 Antibodies

Balb/c mice were immunized with a recombinant human NKp46 extracellulardomain recombinant-Fc protein. Mice received one primo-immunization withan emulsion of 50 μg NKp46 protein and Complete Freund Adjuvant,intraperitoneally, a 2nd immunization with an emulsion of 50 μg NKp46protein and Incomplete Freund Adjuvant, intraperitoneally, and finally aboost with 10 μg NKp46 protein, intravenously. Immune spleen cells werefused 3 days after the boost with X63.Ag8.653 immortalized B cells, andcultured in the presence of irradiated spleen cells.

Primary screen: Supernatant (SN) of growing clones were tested in aprimary screen by flow cytometry using a cell line expressing the humanNKp46 construct at the cell surface. Briefly, for FACS screening, thepresence of reacting antibodies in supernanants was revealed by Goatanti-mouse polyclonal antibody (pAb) labeled with PE.

A selection of antibodies that bound NKp46 were selected, produced asfull-length human IgG antibodies and as bispecific antibodies. Among thevariable regions evaluated for their activity in the context of abispecific molecule in Examples 2-13 were antibodies NKp46-1, -2, -3,-4, -6 and -9 having the respective variable regions shown in Table Bherein.

Part B: Generation of Humanized Anti-Human/Anti-Cynomologus NKp46Antibodies

Antibodies NKp46-1, -2, -3 and -4 having the respective variable regionsshown in Table B herein were produced as humanized antibodies bycomplementary determining region (CDR) grafting of heavy and lightchains having the amino acid sequence shown below. Antibodies wereproduced using CHO cells and tested for binding to human NKp46.

Each of the CDR-grafted antibodies bound with good affinity to humanNKp46. However, none of the CDR-grafted antibodies bound to cynomologusNKp46. Epitopes on human NKp46 were determined (see Example 13); in viewof the possibility that modifications that affect the conformation ofthe antibody variable regions or positioning of the CDRs may permit anepitope shared on cynomolgus NKp46 to be recognized, multiple variantswere prepared for each of the CDR-grafted antibodies for NKp46-1, -2,-3, -4, and -9 were prepared and produced using CHO cells, and testedfor binding to cynomolgus NKp46. For each of antibody NKp46-1, -2, -3,-4, and -9, one more variants were identified that permitted therecognition with good affinity of an epitope shared by human andcynomolgus NKp46 binding. Based on 3D modelling studies, different heavyand light chain variable regions were designed that included NKp46-1CDRs and human frameworks, produced as human IgG1 antibodies, and testedfor binding to cynomolgus NKp46.

Antibody NKp46-1

Based on 3D modelling studies, different heavy and light chain variableregions were designed that included NKp46-1 CDRs and human frameworks,produced as human IgG1 antibodies, and tested for binding to cynomolgusNKp46. Two combinations of heavy and light chains were able to bind toboth human and cynomolgus NKp46: the heavy chain variable region “H1”and the heavy chain “H3”, in each case combined with the light chain“L1”. These cross-binding variable regions included, for the heavy chainvariable region: the NKp46-1 heavy chain CDRs (shown below, underlined),human IGHV1-69*06 gene framework 1, 2 and 3 regions and a human IGHJ6*01gene framework 4 region. The light chain variable region: the NKp46-1light chain CDRs (shown below, underlined), human IGKV1-33*01 geneframework 1, 2 and 3 regions and a human IGKJ4*01 gene framework 4region. CDRs were chosen according to Kabat numbering. The H1, H3 and L1chain had the specific amino acid substitutions (shown in bold andunderlining below). L1 had a phenylalanine at Kabat light chain residue87. H1 had a tyrosine at Kabat heavy chain residue 27 and a lysine andalanine at Kabat residues 66 and 67, respectively. H3 additionally had aglycine at Kabat residue 37, an isoleucine at Kabat residue 48, and aphenylalanine at Kabat residue 91.

NKp46-1: “H1” Heavy Chain Variable Region

(SEQ ID NO: 199) QVQLVQSGAEVKKPGSSVKVSCKASG Y TFSDYVINWVRQAPGQGLEWMGEIYPGSGTNYYNEKFKAKA TITADKSTSTAYMELSSLRSEDTAVYYCARRGRYGLYAMDYWGQGTTVTVSSNKp46-1: “H3” Heavy Chain Variable Region

(SEQ ID NO: 200) QVQLVQSGAEVKKPGSSVKVSCKASG Y TF TDYVINW G RQAPGQGLEW IG EIYPGSGTNYYNEKFKAKA TITADKSTSTAYMELSSLRSEDTAVY F CARRGRYGLYAMDYWGQGTTVTVSSNKp46-1: “L1” Light Chain Variable Region

(SEQ ID NO: 201) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATY F CQQGNTRPWTF GGGTKVEIK

Antibody NKp46-2

Based on 3D modelling studies, different heavy and light chain variableregions were designed that included NKp46-2 CDRs and human frameworks,produced as human IgG1 antibodies, and tested for binding to cynomolgusNKp46. Three combinations of heavy and light chains were able to bind toboth human and cynomolgus NKp46: the heavy chain variable regions “H1”,“H2 and “H3”, in each case combined with the light chain “L1”.Interestingly, the H1L1 furthermore had an improved binding affinitycompared to the parental NKp46-2 antibody having the VH and VL of SEQ IDNOS: 5 and 6. These cross-binding variable regions included, for theheavy chain variable region: the NKp46-2 heavy chain CDRs (shown below,underlined), human IGHV4-30-4*01 gene framework 1, 2 and 3 regions and ahuman IGHJ1*01 gene framework 4 region. The light chain variable region:the NKp46-2 light chain CDRs (shown below, underlined), humanIGKV1-39*01 gene framework 1, 2 and 3 regions and a human IGKJ4*01 geneframework 4 region. CDRs were chosen according to Kabat numbering. TheL1, H1, H2 and H3 chains had the specific amino acid substitutions(shown in bold and underlining below). L1 had a valine at Kabat lightchain residue 48. H1 had a tyrosine at Kabat heavy chain residue 27 andan arginine at Kabat residue 71. H2 additionally had a methionine atKabat residue 48 and an isoleucine at Kabat residue 67. H3 additionallyhad a threonine at Kabat residue 31.

NKp46-2: “H1” Heavy Chain Variable Region

(SEQ ID NO: 202) QVQLQESGPGLVKPSQTLSLTCTVSG Y SISSDYAWNWIRQPPGKGLEWIGYITYSGSTSYNPSLESRVTIS R DTSKNQFSLKLSSVTAADTAVYYCARGGYYGSSWGVFAYWGQGTLVTVSSNKp46-2: “H2” Heavy Chain Variable Region

(SEQ ID NO: 203) QVQLQESGPGLVKPSQTLSLTCTVSG Y SISSDYAWNWIRQPPGKGLEW MGYITYSGSTSYNPSLESR I TIS R DTSKNQFSLKLSSVTAADTAVYYCARGGYYGSSWGVFAYWGQGTLVTVSSNKp46-2: “H3” Heavy Chain Variable Region

(SEQ ID NO: 204) QVQLQESGPGLVKPSQTLSLTCTVSG Y SIT SDYAWNWIRQPPGKGLEW M GYITYSGSTSYNPSLESR I TIS R DTSKNQFSLKLSSVTAADTAVYYCARGGYYGSSWGVFAYWGQGTLVTVSSNKp46-2: “L1” Light Chain Variable Region

(SEQ ID NO: 205) DIQMTQSPSSLSASVGDRVTITCRVSENTYSYLAWYQQKPGKAPKLL V YNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPWTF GGGTKVEIK

Antibody NKp46-3

Based on 3D modelling studies, different heavy and light chain variableregions were designed that included NKp46-3 CDRs and human frameworks,produced as human IgG1 antibodies, and tested for binding to cynomolgusNKp46. Three combinations of heavy and light chains were able to bind toboth human and cynomolgus NKp46: the heavy chain variable regions “H1”,“H3 and “H4”, in each case combined with the light chain “L1”.Interestingly, the H3L1 furthermore had an improved binding affinitycompared to the parental NKp46-3 antibody having the VH and VL of SEQ IDNOS: 7 and 8. These cross-binding variable regions included, for theheavy chain variable region: the NKp46-3 heavy chain CDRs (shown below,underlined), human IGHV1-69*02 gene framework 1, 2 and 3 regions and ahuman IGHJ6*01 gene framework 4 region. The light chain variable region:the NKp46-3 light chain CDRs (shown below, underlined), framework 1, 2and 3 regions created by a mosaic approach using FR1 and FR2 fromIGKV3-15 and FR3 from IGKV3-11, and a human IGKJ2*01 gene framework 4region. CDRs were chosen according to Kabat numbering. The L1, H1, H3and H4 chains had the specific amino acid substitutions (shown in boldand underlining below). L1 had a lysine at Kabat light chain residue 49.H1 had a tyrosine at Kabat heavy chain residue 27. H3 additionally had aisoleucine at Kabat residue 48 and an alanine at Kabat residue 67. H4additionally had a leucine at Kabat residue 69.

NKp46-3: “H1” Heavy Chain Variable Region

(SEQ ID NO: 206) QVQLVQSGAEVKKPGSSVKVSCKASG Y TFSEYTMHWVRQAPGQGLEWMGGISPNIGGTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR RGGSFDYWGQGTTVTVSSNKp46-3: “H3” Heavy Chain Variable Region

(SEQ ID NO: 207) QVQLVQSGAEVKKPGSSVKVSCKASG Y TFSEYTMHWVRQAPGQGLEW I GGISPNIGGTSYNQKFKGR A TITADKSTSTAYMELSSLRSEDTAVYYCAR RGGSFDYWGQGTTVTVSSNKp46-3: “H4” Heavy Chain Variable Region

(SEQ ID NO: 208) QVQLVQSGAEVKKPGSSVKVSCKASG Y TFSEYTMHWVRQAPGQGLEW I GGISPNIGGTSYNQKFKGR A T L TADKSTSTAYMELSSLRSEDTAVYYCAR RGGSFDYWGQGTTVTVSSNKp46-3: “L1” Light Chain Variable Region

(SEQ ID NO: 209) EIVMTQSPATLSVSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIKYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPL TFGQGTKLEIK

Antibody NKp46-4

Based on 3D modelling studies, different heavy and light chain variableregions were designed that included NKp46-4 CDRs and human frameworks,produced as human IgG1 antibodies, and tested for binding to cynomolgusNKp46. The heavy chain variable region “H1” combined with the lightchain “L2” was able to bind to both human and cynomolgus NKp46 as wellas the parental antibody. Two other antigen binding regions (onecomposed of “L2” and “H2”, and one composed of “L2” and “H3”) were ableto bind cynomolgus NKp46 at intermediate levels, although with anaffinity that was 10-fold lower that for human NKp46. Thesecross-binding variable regions included, for the heavy chain variableregion: the NKp46-4 heavy chain CDRs (shown below, underlined), humanframework 1, 2 and 3 regions designed using a mosaic approach using FR1from IGHV1646,0 FR3 from IGHV1-69, and a common FR2 (IGHV1-46 andIGHV1-69 share the same FR2), and a human IGHJ6*01 gene framework 4region. The light chain variable region: the NKp46-4 light chain CDRs(shown below, underlined), framework 1, 2 and 3 regions from IGKV1-NL1,and a human IGKJ4*01 gene framework 4 region. CDRs were chosen accordingto Kabat numbering. The L2, H1, H2 and H3 chains had the specific aminoacid substitutions (shown in bold and underlining below). L2 had aphenylalanine at Kabat light chain residue 36 and a valine at Kabatresidue 48. H1 had a threonine at Kabat heavy chain residue 30 and anisoleucine at Kabat residue 48 and a valine at Kabat residue 93. H2additionally had a threonine at Kabat residue 67. H3 additionally had aleucine at Kabat residue 69.

NKp46-4: “H1” Heavy Chain Variable Region

(SEQ ID NO: 210) QVQLVQSGAEVKKPGASVKVSCKASGYTF TSFTMHWVRQAPGQGLEW I GYINPSSGYTEYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYC V R GSSRGFDYWGQGTLVTVSSNKp46-4: “H2” Heavy Chain Variable Region

(SEQ ID NO: 211) QVQLVQSGAEVKKPGASVKVSCKASGYTF TSFTMHWVRQAPGQGLEW I GYINPSSGYTEYNQKFKDR T TITADKSTSTAYMELSSLRSEDTAVYYC V RGSSRGFDYWGQGTLVTVSSNKp46-4: “H3” Heavy Chain Variable Region

(SEQ ID NO: 212) QVQLVQSGAEVKKPGASVKVSCKASGYTF TSFTMHWVRQAPGQGLEW I GYINPSSGYTEYNQKFKDR T T L TADKSTSTAYMELSSLRSEDTAVYYC V RGSSRGFDYWGQGTLVTVSSNKp46-4: “L2” Light Chain Variable Region

(SEQ ID NO: 213) DIQMTQSPSSLSASVGDRVTITCRASENTYSNLAW F QQKPGKAPKLL V YAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPRTF GGGTKVEIK

Antibody NKp46-9

Based on 3D modelling studies, different heavy and light chain variableregions were designed that included NKp46-9 CDRs and human frameworks,produced as human IgG1 antibodies, and tested for binding to cynomolgusNKp46. Heavy chain variable region “H2”, when combined with either lightchain variable region “L1” or “L2”, and heavy chain variable region “H3”when combined with either light chain variable region “L1” or “L2”, wereable to bind to both human and cynomolgus NKp46 with greater affinitythat the parental antibody (having the VH and VL of SEQ ID NOS: 13 and14. The “H1” chain was not able to bind cynomolgus NKp46 with any of thelight chains tested but bound well to human NKp46. These cross-bindingvariable regions included, for the heavy chain variable region: theNKp46-9 heavy chain CDRs (shown below, underlined), human framework 1, 2and 3 regions from IGHV4-30-4*01, and a human IGHJ6*01 gene framework 4region. The light chain variable region: the NKp46-9 light chain CDRs(shown below, underlined), framework 1, 2 and 3 regions fromIGKV1-39*01, and a human IGKJ2*01 gene framework 4 region. CDRs werechosen according to Kabat numbering. The L1, L2, H1, H2 and H3 chainshad the specific amino acid substitutions (shown in bold and underliningbelow). L1 had a cysteine at Kabat light chain residue 36. L2additionally had a valine at Kabat residue 48. H1 had an arginine atKabat heavy chain residue 71. H2 additionally had a tyrosine at Kabatresidue 27. H3 additionally had a methionine at Kabat residue 48 and anisoleucine at Kabat residue 67.

NKp46-9: “H1” Heavy Chain Variable Region

(SEQ ID NO: 214) QVQLQESGPGLVKPSQTLSLTCTVSGGSISSDYAWNWIRQPPGKGLEWIGYITYSGSTNYNPSLKSRVTIS R DTSKNQFSLKLSSVTAADTAVYYCARCWDYALYAMDCWGQGTTVTVSSNKp46-9: “H2” Heavy Chain Variable Region

(SEQ ID NO: 215) QVQLQESGPGLVKPSQTLSLTCTVSG Y SISSDYAWNWIRQPPGKGLEWIGYITYSGSTNYNPSLKSRVTIS R DTSKNQFSLKLSSVTAADTAVYYCARCWDYALYAMDCWGQGTTVTVSSNKp46-9: “H3” Heavy Chain Variable Region

(SEQ ID NO: 216) QVQLQESGPGLVKPSQTLSLTCTVSG Y SISSDYAWNWIRQPPGKGLEW M GYITYSGSTNYNPSLKSRITIS R DTSKNQFSLKLSSVTAADTAVYYCARCWDYALYAMDCWGQGTTVTVSSNKp46-9: “L1” Light Chain Variable Region

(SEQ ID NO: 217) DIQMTQSPSSLSASVGDRVTITCRTSENTYSYLAW C QQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYDTPLTF GQGTKLEIKNKp46-9: “L2” Light Chain Variable Region

(SEQ ID NO: 218) DIQMTQSPSSLSASVGDRVTITCRTSENTYSYLAW C QQKPGKAPKLL V YNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYDTPLTF GQGTKLEIK

Example 2 Identification of a Bispecific Antibody Format that Binds FcRnbut not FcγR for Targeting Effector Cell Receptors

The aim of this experiment was to develop a new bispecific proteinformat that places an Fc domain on a polypeptide together with ananti-NKp46 binding domain and an anti-target antigen binding domain. Thebispecific protein binds to NKp46 monovalently via its anti-NKp46binding domain. The monomeric Fc domain retains at least partial bindingto the human neonatal Fc receptor (FcRn), yet does not substantiallybind human CD16 and/or other human Fcγ receptors. Consequently, thebispecific protein will not induce Fcγ-mediated (e.g. CD16-mediated)target cell lysis.

Example 2-1 Construction and Binding Analysis ofAnti-CD19-IgG1-Fcmono-Anti-CD3

Since no anti-NKp46 bispecific antibody has been produced that couldindicate whether such a protein could be functional, CD3 was used as amodel antigen in place of NKp46 in order to investigate thefunctionality of a new monovalent bispecific protein format prior totargeting NK cells via NKp46.

A bispecific Fc-based on a scFv specific for tumor antigen CD19(anti-CD19 scFv) and a scFV specific for activating receptor CD3 on a Tcell (anti-CD3 scFv) was used to assess FcRn binding and CD19-bindingfunctions of a new monomeric bispecific polypeptide format. The domainarrangement of the final polypeptide is referred to as the “F1” format(the star in the CH2 domain indicates an optional N297S mutation, notincluded in the polypeptide tested here).

A bispecific monomeric Fc-containing polypeptide was constructed basedon an scFv specific for the tumor antigen CD19 (anti-CD19 scFv) and anscFV specific for an activating receptor CD3 on a T cell (anti-CD3scFv). The CH3 domain incorporated the mutations (EU numbering) L351K,T366S, P395V, F405R, T407A and K409Y. The polypeptide has domainsarranged as follows: anti-CD19-CH2-CH3-anti-CD3. DNA sequence coding fora CH3/VH linker peptide having the amino acid sequence STGS was designedin order to insert a specific SalI restriction site at the CH3-VHjunction.

The CH3 domain incorporated the mutations (EU numbering) L351K, T366S,P395V, F405R, T407A and K409Y. The CH2 domain was a wild-type CH2. DNAand amino acid sequences for the monomeric CH2-CH3 Fc portion and theanti-CD19 are shown below.

The light chain and heavy chain DNA and amino acid sequencescorresponding to the anti-CD19 scFv were as follows:

Sequence SEQ ID NO Anti-CD19-VK DNA 113 Anti-CD19-VK amino acid 114Anti-CD19-VH DNA 115 Anti-CD19-VH amino acid 116

The DNA sequences for the monomeric CH2-CH3 Fc portion and finalbispecific IgG1-Fcmono polypeptide (the last K was removed in thatconstruct) is shown in SEQ ID NO: 117. The amino acid sequence is shownin SEQ ID NO: 2. The Anti-CD19-F1-Anti-CD3 complete sequence (matureprotein) is shown in SEQ ID NO: 118.

Cloning and Production of the Recombinant Proteins

Coding sequences were generated by direct synthesis and/or by PCR. PCRwere performed using the PrimeSTAR MAX DNA polymerase (Takara, #R045A)and PCR products were purified from 1% agarose gel using the NucleoSpingel and PCR clean-up kit (Macherey-Nagel, #740609.250). Once purifiedthe PCR product were quantified prior to the In-Fusion ligation reactionperformed as described in the manufacturer's protocol (ClonTech,#ST0345). The plasmids were obtained after a miniprep preparation run onan EVO200 (Tecan) using the Nucleospin 96 plasmid kit (Macherey-Nagel,#740625.4). Plasmids were then sequenced for sequences confirmationbefore to transfecting the CHO cell line.

CHO cells were grown in the CD-CHO medium (Invitrogen) complemented withphenol red and 6 mM GlutaMax. The day before the transfection, cells arecounted and seeded at 175.000 cells/ml. For the transfection, cells(200.000 cells/transfection) are prepared as described in the AMAXA SFcell line kit (AMAXA, #V4XC-2032) and nucleofected using the DS137protocol with the Nucleofector 4D device. All the tranfections wereperformed using 300 ng of verified plasmids. After transfection, cellsare seeded into 24 well plates in pre-warmed culture medium. After 24H,hygromycine B was added in the culture medium (200 μg/ml). Proteinexpression is monitored after one week in culture. Cells expressing theproteins are then sub-cloned to obtain the best producers. Sub-cloningwas performed using 96 flat-bottom well plates in which the cells areseeded at one cell per well into 200 μl of culture medium complementedwith 200 μg/ml of hygromycine B. Cells were left for three weeks beforeto test the clone's productivity.

Recombinant proteins which contain a IgG1-Fc fragment are purified usingProtein-A beads (-rProteinA Sepharose fast flow, GE Healthcare, ref.:17-1279-03). Briefly, cell culture supernatants were concentrated,clarified by centrifugation and injected onto Protein-A columns tocapture the recombinant Fc containing proteins. Proteins were eluted atacidic pH (citric acid 0.1M pH3), immediately neutralized using TRIS-HCLpH8.5 and dialyzed against 1×PBS. Recombinant scFv which contain a “sixhis” tag were purified by affinity chromatography using Cobalt resin.Other recombinant scFv were purified by size exclusion chromatography(SEC).

Example 2-2: Binding Analysis of Anti-CD19-IgG1-Fcmono-Anti-CD3 to B221,JURKAT, HUT78 and CHO Cell Lines

Cells were harvested and stained with the cell supernatant of theanti-CD19-F1-anti-CD3 producing cells during 1 H at 4° C. After twowashes in staining buffer (PBS1X/BSA 0.2%/EDTA 2 mM), cells were stainedfor 30 min at 4° C. with goat anti-human (Fc)-PE antibody (IM0550Beckman Coulter—1/200). After two washes, stainings were acquired on aBD FACS Canto II and analyzed using the FlowJo software.

CD3 and CD19 expression were also controlled by flow cytometry: Cellswere harvested and stained in PBS1X/BSA 0.2%/EDTA 2 mM buffer during 30min at 4° C. using 5 μl of the anti-CD3-APC and 5 μl of theanti-CD19-FITC antibodies. After two washes, stainings were acquired ona BD FACS Canto II and analyzed using the FlowJo software.

The Anti-CD19-F1-Anti-CD3 protein binds to the CD3 cell lines (HUT78 andJURKAT cell lines) and the CD19 cell line (B221 cell line) but not tothe CHO cell line used as a negative control.

Example 2-3 T- and B-Cell Aggregation by Purified Anti-CD19-F1-Anti-CD3

Purified Anti-CD19-F1-Anti-CD3 was tested in a T/B cell aggregationassay to evaluate whether the antibody is functional in bringingtogether CD19 and CD3 expressing cells.

Results are shown in FIG. 1 . The top panel shows thatAnti-CD19-F1-Anti-CD3 does not cause aggregation in the presence of B221(CD19) or JURKAT (CD3) cell lines, but it does cause aggregation ofcells when both B221 and JURKAT cells are co-incubated, illustratingthat the bispecific antibody is functional. The lower panel showscontrol without antibody.

Example 2-4 Binding of Bispecific Monomeric Fc Polypeptide to FcRn

Affinity Study by Surface Plasmon Resonance (SPR)

Biacore T100 General Procedure and Reagents

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments Acetate Buffer (50 mMAcetate pH5.6, 150 mM NaCl, 0.1% surfactant p20) and HBS-EP+ (Biacore GEHealthcare) served as running buffer and regeneration bufferrespectively. Sensorgrams were analyzed with Biacore T100 Evaluationsoftware. Recombinant mouse FcRn was purchase from R&D Systems.

Immobilization of FcRn

Recombinant FcRn proteins were immobilized covalently to carboxyl groupsin the dextran layer on a Sensor Chip CM5. The chip surface wasactivated with EDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GEHealthcare)). FcRn proteins were diluted to 10 μg/ml in coupling buffer(10 mM acetate, pH 5.6) and injected until the appropriateimmobilization level was reached (i.e. 2500 RU). Deactivation of theremaining activated groups was performed using 100 mM ethanolamine pH 8(Biacore GE Healthcare).

Affinity Study

Monovalent affinity study was done following the Single Cycle Kinetic(SCK) protocol. Five serial dilutions of soluble analytes (antibodiesand bi-specific molecules) ranging from 41.5 to 660 nM were injectedover the FcRn (without regeneration) and allowed to dissociate for 10min before regeneration. For each analyte, the entire sensorgram wasfitted using the 1:1 SCK binding model.

Results

Anti-CD19-F1-Anti-CD3 having its CH2-CH3 domains placed between twoantigen binding domains, here two scFv, was evaluated to assess whethersuch bispecific monomeric Fc protein could retain binding to FcRn andthereby have improved in vivo half-lives compared to conventionbispecific antibodies. Results showed that FcRn binding was retained,the model suggesting 1:1 ratio (1 FcRn for each monomeric Fc) instead of2:1 ration (2 FcRn for each antibody) for a regular IgG.

Affinity was evaluated using SPR, in comparison to a chimeric fulllength antibody having human IgG1 constant regions. The monomeric Fcretained significant monomeric binding to FcRn (monomeric Fc: affinityof KD=194 nM; full length antibody with bivalent binding: avidity ofKD=15.4 nM).

Example 3 Construction of Anti-CD19×Anti-NKp46 Bispecific Monomeric FcDomain Polypeptides

It was unknown what activating receptors on NK cells would contribute tothe lysis of target cells, and moreover since anti-NKp46 antibodies mayblock NKp46, it was further unknown whether cytotoxicity could bemediated by NKp46. We therefore investigated whether the bispecificprotein format could induce NKp46 triggering, and whether it wouldinduce NKp46 agonism in the absence of target cells, which could lead toinappropriate NK activation distant from the target and/or decreasedoverall activity toward target cells.

A new bispecific protein format was developed as a single chain proteinwhich binds to FcRn but not FcγR. Additionally, multimeric proteins thatcomprise two or three polypeptide chains, wherein the Fc domain remainsmonomeric, were developed that are compatible for use with antibodyvariable regions that do not maintain binding to their target whenconverted to scFv format. The latter formats can be used convenientlyfor antibody screening; by incorporating at least one binding region asa F(ab) structure, any anti-target (e.g. anti-tumor) antibody variableregion can be directly expressed in a bispecific construct as the F(ab)format within the bispecific protein and tested, irrespective of whetherthe antibody would retain binding as an scFv, thereby simplifyingscreening and enhancing the number of antibodies available. Theseformats in which the Fc domain remains monomeric have the advantage ofmaintaining maximum conformational flexibility and as shown infra maypermit optimal binding to NKp46 or target antigens.

Different constructs were made for use in the preparation of bispecificantibodies using the variable domains from the scFv specific for tumorantigen CD19 described in Example 2-1, and different variable regionsfrom antibodies specific for the NKp46 receptor identified in Example 1.A construct was also made using as the anti-NKp46 the variable regionsfrom a commercially available antibody Bab281 (mIgG1, availablecommercially from Beckman Coulter, Inc. (Brea, Calif., USA) (see alsoPessino et al, J. Exp. Med, 1998, 188 (5): 953-960 and Sivori et al, EurJ Immunol, 1999. 29:1656-1666) specific for the NKp46 receptor.

In order for the Fc domain to remain monomeric in single chainpolypeptides or in multimers in which only one chain had an Fc domain,CH3-CH3 dimerization was prevented through two different strategies: (1)through the use of CH3 domain incorporating specific mutations (EUnumbering), i.e., L351K, T366S, P395V, F405R, T407A and K409Y; or (2)through the use of a tandem CH3 domain in which the tandem CH3 domainsare separated by a flexible linker associated with one another, whichprevents interchain CH3-CH3 dimerization. The DNA and amino acidsequences for the monomeric CH2-CH3 Fc portion containing theabove-identified point mutations were the same as in Example 2-1. TheDNA and amino acid sequences for the monomeric CH2-CH3-linker-CH3 Fcportion with tandem CH3 domains are shown in FIGS. 2A-2D.

The light chain and heavy chain DNA and amino acid sequences for theanti-CD19 scFv were also the same as in Example 2-1. Proteins werecloned, produced and purified as in Example 2-1. Shown below are thelight chain and heavy chain DNA and amino acid sequences for differentanti-NKp46 scFvs.

TABLE 1 Amino acid sequences of different anti-NKp46 scFvs scFvanti-NKp46 scFV sequence (VHVK)/-stop NKp46-1STGSQVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEIYPGSGTNYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRYGLYAMDYWGQGTSVTVSSVEGGSGGSGGSGGSGGVDDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGTKLEIK- (SEQ ID NO: 119) NKp46-2STGSEVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTSYNPSLESRISITRDTSTNQFFLQLNSVTTEDTATYYCARGGYYGSSWGVFAYWGQGTLVTVSAVEGGSGGSGGSGGSGGVDDIQMTQSPASLSASVGETVTITCRVSENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPWTFGGGTKLEIK- (SEQ ID NO: 120) NKp46-3STGSEVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGISPNIGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGGSFDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLE LK- (SEQ ID NO: 121)NKp46-4 STGSQVQLQQSAVELARPGASVKMSCKASGYTFTSFTMHWVKQRPGQGLEWIGYINPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLDSLTSDDSAVYYCVRGSSRGFDYWGQGTLVTVSAVEGGSGGSGGSGGSGGVDDIQMIQSPASLSVSVGETVTITCRASENIYSNLAWFQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGIYYCQHFWGTPRTFGGGTKLEIK- (SEQ ID NO: 122) NKp46-6STGSQVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWMHWAKQRPGQGLEWIGHIHPNSGISNYNEKFKGKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARGGRFDDWGAGTTVTVSSVEGGSGGSGGSGGSGGVDDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFLMYTFGGGTKL EIK- (SEQ ID NO: 123)NKp46-9 STGSDVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARCWDYALYAMDCWGQGTSVTVSSVEGGSGGSGGSGGSGGVDDIQMTQSPASLSASVGETVTITCRTSENIYSYLAWCQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTHFSLKINSLQPEDFGIYYCQHHYDTPLTFGAGTKLELK- (SEQ ID NO: 124) Bab281STGSQIQLVQSGPELQKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTNTGEPTYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYLYYFDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDNIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIK- (SEQ ID NO: 125)

TABLE 2A DNA sequences corresponding to different anti-NKp46 scFvs scFvanti-NKp46 scFV sequences NKp46-1 SEQ ID NO: 126 NKp46-2 SEQ ID NO: 127NKp46-3 SEQ ID NO: 128 NKp46-4 SEQ ID NO: 129 NKp46-6 SEQ ID NO: 130NKp46-9 SEQ ID NO: 131 Bab281 SEQ ID NO: 132Format 1 (F1) (Anti-CD19-IgG1-Fcmono-Anti-NKp46 (scFv))

The domain structure of Format 1 (F1) is shown in FIG. 2A. A bispecificFc-containing polypeptide was constructed based on a scFv specific forthe tumor antigen CD19 (anti-CD19 scFv) and an scFV specific for theNKp46 receptor. The polypeptide is a single chain polypeptide havingdomains arranged (N- to C-termini) as follows: (VK-V_(H))^(anti-CD19)CH2-CH3-(V_(H)-VK)^(anti-NKp46)

A DNA sequence coding for a CH3/VH linker peptide having the amino acidsequence STGS was designed in order to insert a specific SalIrestriction site at the CH3-V_(H) junction. The domain arrangement ofthe final polypeptide in shown in FIG. 2 (the star in the CH2 domainindicates an optional N297S mutation), where the anti-CD3 scFv isreplaced by an anti-NKp46 scFv. The (VK-V_(H)) units include a linkerbetween the V_(H) and VK domains. Proteins were cloned, produced andpurified as in Example 2-1. The amino acid sequences of the bispecificpolypeptides (complete sequence (mature protein)) are shown in thecorresponding SEQ ID NOS listed in the Table 2B below.

TABLE 2B Sequence SEQ ID NO CD19-F1-NKp46-1 133 CD19-F1-NKp46-2 134CD19-F1-NKp46-3 135 CD19-F1-NKp46-4 136 CD19-F1-NKp46-6 137CD19-F1-NKp46-9 138 CD19-F1-Bab281 139Format 2 (F2): CD19-F2-NKp46-3

The domain structure of F2 polypeptides is shown in FIG. 2A. The DNA andamino acid sequences for the monomeric CH2-CH3 Fc portion were as inExample 2-1 and it similarly contains CH3 domain mutations (themutations (EU numbering) L351K, T366S, P395V, F405R, T407A and K409Y.The heterodimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-termini):(VK-V_(H))^(anti-CD19)CH2-CH3-V_(H) ^(anti-NKp46)—CH1and

(2) a second polypeptide chain having domains arranged as follows (N- toC-termini): VK^(anti-NIKP46)-CK.

The (VK-V_(H)) unit was made up of a V_(H) domain, a linker and a VKCunit (i.e. an scFv). As with other formats of the inventive bispecificpolypeptides, the DNA sequence coded for a CH3/VH linker peptide havingthe amino acid sequence STGS designed in order to insert a specific SalIrestriction site at the CH3-VH junction. Proteins were cloned, producedand purified as in Example 2-1. The amino acid sequences for the firstand second chains of the F2 protein are shown in SEQ ID NO: 140 and 141.

Format 3 (F3): CD19-F3-NKp46-3

The domain structure of F3 polypeptides is shown in FIG. 2A. The DNA andamino acid sequences for the CH2-CH3 Fc portion comprised a tandem CH3domain in which the two CH3 domains on the same polypeptide chainassociated with one another, thereby preventing dimerization betweendifferent bispecific proteins.

The single chain polypeptide has domains arranged (N- to C-termini) asfollows: (VK-V_(H))^(anti-CD19) CH2-CH3-CH3-(V_(H)-VK)^(anti-NKp46).

The (VK-V_(H)) units were made up of a V_(H) domain, a linker and a VKunit (scFv). Proteins were cloned, produced and purified as in Example2-1. Bispecific protein was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a high production yield of 3.4 mg/L and thepurified proteins exhibited a simple SEC profile. The amino acidsequence for the F3 protein is shown in SEQ ID NO: 142.

Format 4 (F4): CD19-F4-NKp46-3

The domain structure of F4 polypeptides is shown in FIG. 2A. The DNA andamino acid sequences for the CH2-CH3 Fc portion comprised a tandem CH3domain as in Format F3, and additionally comprise a N297S mutation whichprevents N-linked glycosylation and abolishes FcγR binding. Proteinswere cloned, produced and purified as in Example 2-1. Bispecificproteins was purified from cell culture supernatant by affinitychromatography using prot-A beads and analyzed and purified by SEC. Theprotein showed a good production yield of 1 mg/L and the purifiedproteins exhibited a simple SEC profile. The amino acid sequence for theF4 protein with NKp46-3 variable domains is shown in SEQ ID NO: 143.

Format 8 (F8)

The domain structure of F8 polypeptides is shown in FIG. 2B. The DNA andamino acid sequences for the monomeric CH2-CH3 Fc portion were as inFormat F2 and it similarly contains CH3 domain mutations (the mutations(EU numbering) L351K, T366S, P395V, F405R, T407A and K409Y, as well as aN297S mutation which prevents N-linked glycosylation and moreoverabolishes FcγR binding. Three variants of F8 proteins were produced: (a)one wherein the cysteine residues in the hinge region were left intact(wild-type, referred to as F8A), (b) a second wherein the cysteineresidues in the hinge region were replaced by serine residues (F8B), and(c) a third including a linker sequence GGGSS replacing residuesDKTHTCPPCP in the hinge (F8C). Variants F8B and F8C provided productionadvantages as these versions avoided the formation of homodimers of thecentral chain. This heterotrimer is made up of;

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-termini):V_(H) ^(anti-CD19)-CH1-CH2-CH3-VH^(anti-NKP46)-CK;and

(2) a second polypeptide chain having domains arranged as follows (N- toC-termini): VK^(anti-NKp46)-CH1;

and

-   -   (3) a third polypeptide chain having domains arranged as follows        (N- to C-termini):        VK ^(anti-CD19)-CK.

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a high production yield of 3.7 mg/L (F8C) andthe purified proteins again exhibited a simple SEC profile. The aminoacid sequences of the three chains of the F8 protein (C variant) withNKp46-3 variable regions are shown in SEQ ID NOS: 144, 145 and 146.

Format 9 (F9): CD19-F9-NKp46-3

The F9 polypeptide is a trimeric polypeptide having a centralpolypeptide chain and two polypeptide chains each of which associatewith the central chain via CH1-CK dimerization. The domain structure ofthe trimeric F9 protein is shown in FIG. 2B, wherein the bonds betweenthe CH1 and CK domains are interchain disulfide bonds. The two antigenbinding domains have a F(ab) structure permitting the use of theseantibodies irrespective of whether they remain functional in a scFvformat. The DNA and amino acid sequences for the CH2-CH3 Fc portioncomprise a tandem CH3 domain as in Format F4 and comprise a CH2 domaincomprising a N297S substitution. Three variants of F9 proteins wereproduced: (a) a first wherein the cysteine residues in the hinge regionleft intact (wild-type, referred to as F9A), (b) a second wherein thecysteine residues in the hinge region were replaced by serine residues(F9B), and (c) a third containing a linker sequence GGGSS which replacesresidues DKTHTCPPCP in the hinge (F9C). Variants F9B and F9C providedadvantages in production by avoiding the formation of homodimers of thecentral chain. The heterotrimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-termini):V_(H) ^(anti-CD19)-CH1-CH2-CH3-CH3-V_(H) ^(anti-NKp46)-CKand

(2) a second polypeptide chain having domains arranged as follows (N- toC-termini): VK ^(anti-1\1″46)-CH1

and

(3) a third polypeptide chain having domains arranged as follows (N- toC-termini):VK ^(anti-CD19)-CK.

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a high production yield of 8.7 mg/L (F9A) and3.0 mg/L (F9B), and the purified proteins again exhibited a simple SECprofile.

The amino acid sequences of the three chains of the F9 protein variantF9A are shown in the SEQ ID NOS: 147, 148 and 149. The amino acidsequences of the three chains of the F9 protein variant F9B are shown inthe SEQ ID NOS: 150, 151 and 152. The amino acid sequences of the threechains of the F9 protein variant F9C are shown in the SEQ ID NOS: 153,154 and 155.

Format 10 (F10): CD19-F10-NKp46-3

The F10 polypeptide is a dimeric protein having a central polypeptidechain and a second polypeptide chain which associates with the centralchain via CH1-CK dimerization. The domain structure of the dimeric F10protein is shown in FIG. 2B wherein the bonds between the CH1 and CKdomains are interchain disulfide bonds. One of the two antigen bindingdomains has a Fab structure, and the other is a scFv. The DNA and aminoacid sequences for the CH2-CH3 Fc portion comprise a tandem CH3 domainas shown in Format F4 and comprise a CH2 domain containing a N297Ssubstitution. Three variants of F10 proteins were also produced: (a) afirst wherein the cysteine residues in the hinge region were left intact(wild-type, referred to as F10A), (b) a second wherein the cysteineresidues in the hinge region were replaced by serine residues (F10B),and (c) a third containing a linker sequence GGGSS replacing residuesDKTHTCPPCP in the hinge (F100). Variants F10B and F100 providedadvantages in production as they avoid the formation of homodimers ofthe central chain. The (VK-V_(H)) unit was made up of a V_(H) domain, alinker and a VK unit (scFv). The heterodimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-termini):V_(H) ^(anti-CD19)-CH1-CH2-CH3-CH3-(V_(H)-VK)^(anti-NKp46)and

(2) a second polypeptide chain having domains arranged as follows (N- toC-termini): VK ^(anti-CD19)-CK.

These proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a good production yield of 2 mg/L (F10A) and thepurified proteins again exhibited a simple SEC profile. The amino acidsequences of the two chains of the F10A protein variant are shown in SEQID NOS: 156 (second chain) and 157 (first chain). The amino acidsequences of the two chains of the F10B protein variant are shown in SEQID NOS: 158 (second chain) and 159 (first chain). The amino acidsequences of the two chains of the F10C protein variant are shown in theSEQ ID NOS: 160 (second chain) and 161 (first chain).

Format 11 (F11): CD19-F11-NKp46-3

The domain structure of F11 polypeptides is shown in FIG. 2C. Theheterodimeric protein is similar to F10 except that the structures ofthe antigen binding domains are reversed. One of the two antigen bindingdomains has a Fab-like structure, and the other is a scFv. Theheterodimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-termini):(VK-V_(H))^(anti-CD19)-CH2-CH3-CH3-VH^(anti-NKp46)-CKand

(2) a second polypeptide chain having domains arranged as follows (N- toC-termini): VK ^(anti-NKp46)-CH1.

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a good production yield of 2 mg/L and thepurified proteins similarly exhibited a simple SEC profile. The aminoacid sequences of the two chains of the F11 protein are shown in SEQ IDNO: 162 (chain 1) and SEQ ID NO: 163 (chain 2).

Format 12 (F12): CD19-F12-NKp46-3

The domain structure of the dimeric F12 polypeptides is shown in FIG.2C, wherein the bonds between the CH1 and CK domains are disulfidebonds. The heterodimeric protein is similar to F11 but the CH1 and CKdomains within the F(ab) structure are inversed. The heterodimer is madeup of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-termini):(VK-V_(H))^(anti-CD19)CH2-CH3-CH3-V_(H) ^(anti-NKp46)—CH1and

(2) a second polypeptide chain having domains arranged as follows (N- toC-termini): VK ^(anti-NKp46)-CK.

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins were purified from the cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a good production yield of 2.8 mg/L and thepurified proteins similarly exhibited a simple SEC profile. The aminoacid sequences of the two chains of the F12 protein are shown in SEQ IDNO: 164 (chain 1) and SEQ ID NO: 165 (chain 2).

Format 17 (F17): CD19-F17-NKp46-3

The domain structure of the trimeric F17 polypeptides is shown in FIG.2C, wherein the bonds between the CH1 and CK domains are disulfidebonds. The heterodimeric protein is similar to F9 but the V_(H) and VKdomains, and the CH1 and CK, domains within the C-terminal F(ab)structure are each respectively inversed with their partner. Theheterotrimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-termini):V_(H) ^(anti-CD19)-CH1-CH2-CH3-CH3-VK ^(anti-NKp46)-CH1;and

(2) a second polypeptide chain having domains arranged as follows (N- toC-termini): V_(H) ^(anti-NKp46)-CK;

and

(3) a third polypeptide chain having domains arranged as follows (N- toC-termini):VK ^(anti-CD19)-CK.

Additionally, three variants of F17 proteins were produced: (a) a firstwhere the cysteine residues in the hinge region were left intact(wild-type, referred to as F17A), (b) a second wherein the cysteineresidues in the hinge region were replaced by serine residues (F10B, and(c) a third containing a linker sequence GGGSS which replaces residuesDKTHTCPPCP in the hinge (F17C). Proteins were cloned, produced andpurified as in Example 2-1. The amino acid sequences of the three chainsof the F17B protein are shown in SEQ ID NOS: 166, 167 and 168.

Example 4 Bispecific NKp46 Antibody Formats with Dimeric Fc Domains

New protein constructions with dimeric Fc domains were developed thatshare many of the advantages of the monomeric Fc domain proteins ofExample 3 but bind to FcRn with greater affinity. Different proteinformats were produced that either had low or substantially lack ofbinding to FcγR (including CD16) or which had binding to FcγRs(including CD16), e.g. the binding affinity to human CD16 was within1-log of that of wild-type human IgG1 antibodies, as assessed by SPR(e.g. see methods of Example 16. The different polypeptide formats weretested and compared to investigate the functionality of heterodimericproteins comprising a central chain with a (V_(H)-(CH1/CK)-CH2-CH3-)unit or a (VK-(CH1 or CK)-CH2-CH3-) unit. One of both of the CH3 domainsare fused, optionally via intervening amino acid sequences or domains,to a variable domain(s) (a single variable domain that associates with avariable domain on a separated polypeptide chain, a tandem variabledomain (e.g., an scFv), or a single variable domain that is capable ofbinding antigen as a single variable domain). The two chains associateby CH1-CK dimerization to form disulfide linked dimers, or if associatedwith a third chain, to form trimers.

Different constructs were made for use in the preparation of abispecific antibody using the variable domains DNA and amino acidsequences derived from the scFv specific for tumor antigen CD19described in Example 2-1 and different variable regions from antibodiesspecific for NKp46 identified in Example 1. Proteins were cloned,produced and purified as in Example 2-1. Domains structures are shown inFIGS. 2A-6D.

Format 5 (F5): CD19-F5-NKp46-3

The domain structure of the trimeric F5 polypeptide is shown in FIG. 2D,wherein the interchain bonds between hinge domains (indicated in thefigures between CH1/CK and CH2 domains on a chain) and interchain bondsbetween the CH1 and CK domains are interchain disulfide bonds. Theheterotrimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-termini):V_(H) ^(anti-CD19)-CH1-CH2-CH3-V_(H) ^(anti-NKp46)-CK;and

(2) a second polypeptide chain having domains arranged as follows (N- toC-termini): VK ^(anti-CD19)-CK-CH2-CH3;

and

(3) a third polypeptide chain having domains arranged as follows (N- toC-termini):VK ^(anti-NKp46)-CH1.

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a high production yield of 37 mg/L and thepurified proteins again exhibited a simple SEC profile. The amino acidsequences of the three polypeptide chains are shown in SEQ ID NOS 169(second chain), 170 (first chain) and 171 (third chain).

Format 6 (F6): CD19-F6-NKp46-3

The domain structure of heterotrimeric F6 polypeptides is shown in FIG.2D. The F6 protein is the same as F5, but contains a N297S substitutionto avoid N-linked glycosylation. Proteins were cloned, produced andpurified as in Example 2-1. Bispecific proteins were purified from cellculture supernatant by affinity chromatography using prot-A beads andanalyzed and purified by SEC. The protein showed a high production yieldof 12 mg/L and the purified proteins exhibited a simple SEC profile. Theamino acid sequences of the three polypeptide chains are shown in SEQ IDNOS: 172 (second chain), 173 (first chain) and 174 (third chain).

Format 7 (F7): CD19-F7-NKp46-3

The domain structure of heterotrimeric F7 polypeptides is shown in FIG.2D. The F7 protein is the same as F6, except for cysteine to serinesubstitutions in the CH1 and CK domains that are linked at theirC-termini to Fc domains, in order to prevent formation of a minorpopulation of dimeric species of the central chain with the VK^(anti-NKp46)-CH1 chain. Proteins were cloned, produced and purified asin Example 2-1. Bispecific proteins were purified from the cell culturesupernatant by affinity chromatography using prot-A beads and analyzedand purified by SEC. The protein showed a high production yield of 11mg/L and the purified proteins exhibited a simple SEC profile. The aminoacid sequences of the three polypeptide chains are shown in SEQ ID NOS:175 (second chain), 176 (first chain) and 177 (third chain).

Format 13 (F13): CD19-F13-NKp46-3

The domain structure of the dimeric F13 polypeptide is shown in FIG. 2D,wherein the interchain bonds between hinge domains (indicated betweenCH1/CK and CH2 domains on a chain) and interchain bonds between the CH1and CK domains are interchain disulfide bonds. The heterodimer is madeup of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-termini):V_(H) ^(anti-CD19)-CH1-CH2-CH3-(V_(H)-VK)^(anti-NKp46)and

(2) a second polypeptide chain having domains arranged as follows (N- toC-termini): VK ^(anti-CD19)-CK-CH2-CH3.

The (V_(H)-VK) unit was made up of a V_(H) domain, a linker and a VKunit (scFv).

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins were purified from the cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a high production yield of 6.4 mg/L and thepurified proteins exhibited a simple SEC profile. The amino acidsequences of the two polypeptide chains are shown in SEQ ID NOS: 178(second chain) and 179 (first chain).

Format 14 (F14): CD19-F14-NKp46-3

The domain structure of the dimeric F14 polypeptide is shown in FIG. 2E.The F14 polypeptide is a dimeric polypeptide which shares the structureof the F13 format, but instead of a wild-type Fc domain (CH2-CH3), theF14 bispecific format has CH2 domain mutations N297S to abolish N-linkedglycosylation. Proteins were cloned, produced and purified as in Example2-1. Bispecific proteins were purified from cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a high production yield of 2.4 mg/L and thepurified proteins exhibited a simple SEC profile. The amino acidsequences of the two polypeptide chains are shown in SEQ ID NOS: 180(second chain) and 181 (first chain).

Format 15 (F15): CD19-F15-NKp46-3

The domain structure of the trimeric F15 polypeptides is shown in FIG.2E. The F15 polypeptide is a dimeric polypeptide which shares thestructure of the F6 format, but differs by inversion of the N-terminalV_(H)-CH1 and VK-CK units between the central and second chains.Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins were purified from the cell culture supernatant byaffinity chromatography using prot-A beads and analyzed and purified bySEC. The protein showed a good production yield of 0.9 mg/L and thepurified proteins possessed a simple SEC profile. The amino acidsequences of the three polypeptide chains are shown in SEQ ID NOS: 182(second chain), 183 (first chain) and 184 (third chain).

Format 16 (F16): CD19-F16-NKp46-3

The domain structure of the trimeric F16 polypeptide is shown in FIG.2E. The F16 polypeptide is a dimeric polypeptide which shares thestructure of the F6 format, but differs by inversion of the C-terminalV_(H)-CK and VK-CH1 units between the central and second chains.Proteins were cloned, produced and purified as in Example 2-1. The aminoacid sequences of the three polypeptide chains are shown in SEQ ID NOS:185 (second chain), 186 (first chain) and 187 (third chain).

Format T5 (T5)

The domain structure of a trimeric T5 polypeptide is shown in FIG. 2F.The T5 polypeptide is a trimeric polypeptide which shares the structureof the F5 format, but differs by fusion of an scFv unit at theC-terminus of the third chain (the chain lacking the Fc domain). Thisprotein will therefore have two antigen binding domains for antigens ofinterest, and one for NKp46, and will bind CD16 via its Fc domain.Proteins were cloned, produced and purified as in Example 2-1. The T5protein had two antigen binding domains that bind human CD20,originating from different antibodies (and binding to different epitopeson CD20). The first anti-CD20 ABD contained the V_(H) and V_(L) of theparent antibody GA101 (GAZYVA®, Gazyvaro®, obinutuzumab, RochePharmaceuticals). The second anti-CD20 ABD contained the V_(H) and V_(L)of the parent antibody rituximab (Rituxan®, Mabthera®, RochePharmaceuticals). The third antigen binding domain binds human NKp46.The amino acid sequences of the three chains of the T5 protein are shownbelow (Rituximab sequences are in bold and underlined, anti-GA101sequences are underlined, anti-NKp46 sequences are in italics).

GA101-T5-Ritux-NKp46

Polypeptide 1 (SEQ ID NO: 188)QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGST GSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-

Polypeptide 2 (SEQ ID NO: 189)DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-

Polypeptide 3: (SEQ ID NO: 190)QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGG TKLEIKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHGGSSSEVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGISPNIGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGGSFDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTF GAGTKLELK-

Format T6 (T6)

The domain structure of the trimeric T6 polypeptide is shown in FIG. 2F.The T6 polypeptide is a trimeric polypeptide which shares the structureof the F6 format, but differs by the fusion of an scFv unit at theC-terminus of the third chain (the chain lacking the Fc domain). Thistrimeric protein contains two antigen binding domains for antigens ofinterest, and one for NKp46, and does not bind CD16 via its Fc domaindue to the N297 substitution. Proteins were cloned, produced andpurified as in Example 2-1. The T6 protein contains two antigen bindingdomains that bind human CD20. The first anti-CD20 ABD comprises theV_(H) and V_(L) of the parent antibody GA101 and the second anti-CD20ABD comprises the V_(H) and V_(L) of rituximab. The amino acid sequencesof the three chains of the T6 proteins are shown in SEQ ID NOS: 191, 192and 193.

Format T98 (T98)

The domain structure of the trimeric T9B polypeptide is shown in FIG.2F. The T9B polypeptide is a trimeric polypeptide which shares thestructure of the F9 format (F9B variant), but differs by the fusion ofan scFv unit at the C-terminus of the free CH1 domain (on the thirdchain). This protein contains two antigen binding domains for antigen ofinterest, and one for NKp46, but will not bind CD16 via its Fc domaindue to the monomeric Fc domain and/or the N297 substitution. Trimericproteins as above described were cloned, produced and purified as inExample 2-1. The T9B protein had two antigen binding domains that bindhuman CD20. The first anti-CD20 ABD contained the V_(H) and V_(L) of theparent antibody GA101 and the second anti-CD20 ABD contained the V_(H)and V_(L) of the parent antibody rituximab. The amino acid sequences ofthe three chains of the T9B proteins are shown below.

GA101-T9B-Ritux-NKp46

Polypeptide 2: (SEQ ID NO: 195)DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC-

Polypeptide 1: (SEQ ID NO: 194)QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSARTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C-

Polypeptide 3 (SEQ ID NO: 196):QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHGGSSSEVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGISPNIGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGGSFDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTF GAGTKLELK-

Format T11 (T1): CD19-T11-NKp46-3

The domain structure of the dimeric T11 polypeptide is shown in FIG. 2F.The T11 polypeptide is a trimeric polypeptide which shares the structureof the F11 format, but differs by the fusion of an scFv unit at theC-terminus of the free CH1 domain. This dimeric protein contains twoantigen binding domains for antigen of interest, and one for NKp46, anddoes not bind CD16 via its Fc domain due to the monomeric Fc domainand/or the N297 substitution. Proteins were cloned, produced andpurified as in Example 2-1. The T11 protein contains two antigen bindingdomains that bind human CD20. The first anti-CD20 ABD contained theV_(H) and V_(L) of the parent antibody GA101 and the second anti-CD20ABD contained the V_(H) and V_(L) of rituximab. The amino acid sequencesof the two chains of the T11 protein are shown below.

GA101-T11-Ritux-NKp46

Polypeptide 1 (SEQ ID NO: 197):DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVROAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSARTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-

Polypeptide 2 (SEQ ID NO: 198):QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHGGSSSEVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGISPNIGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGGSFDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTF GAGTKLELK-

Example 5 NKp46 Binding Affinity by Bispecific Proteins by SurfacePlasmon Resonance (SPR)

Biacore T100 General Procedure and Reagents

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments HBS-EP+(Biacore GEHealthcare) and NaOH 10 mM served as running buffer and regenerationbuffer respectively. Sensorgrams were analyzed with Biacore T100Evaluation software. Protein-A was purchase from (GE Healthcare). HumanNKp46 recombinant proteins were cloned, produced and purified at InnatePharma.

Immobilization of Protein-A

Protein-A proteins were immobilized covalently to carboxyl groups in thedextran layer on a Sensor Chip CM5. The chip surface was activated withEDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl) carbodiimidehydrochlorideand N-hydroxysuccinimide (Biacore GE Healthcare)). Protein-A was dilutedto 10 μg/ml in coupling buffer (10 mM acetate, pH 5.6) and injecteduntil the appropriate immobilization level was reached (i.e. 2000 RU).Deactivation of the remaining activated groups was performed using 100mM ethanolamine pH 8 (Biacore GE Healthcare).

Binding Study

The bispecific proteins were first tested in Format F1 described inExample 2 having different anti-NKp46 variable regions from NKp46-1,NKp46-2, NKp46-3 or NKp46-4 antibodies. Antibodies were next tested asdifferent formats F3, F4, F5, F6, F9, F10, F11, F13, F14 having theanti-NKp46 variable regions from the NKp46-3 antibody, and compared tothe NKp46-3 antibody as a full-length human IgG1.

Bispecific proteins at 1 μg/mL were captured onto Protein-A chip andrecombinant human NKp46 proteins were injected at 5 μg/mL over capturedbispecific antibodies. For blank subtraction, cycles were performedagain replacing NKp46 proteins with running buffer.

The Bab281 antibody was separately tested for binding to NKp46 by SPR,and additionally by flow cytometry using a cell line expressing thehuman NKp46 construct at the cell surface. For FACS screening, thepresence of reacting antibodies in supernanants was revealed by Goatanti-mouse polyclonal antibody (pAb) labeled with PE. SPC and FACSresults showed that the Bab281 based antibody did not bind the NKp46cell line or NKp46-Fc proteins. Bab281 lost binding to its target whenpresented in the bispecific format.

Affinity Study

Monovalent affinity study was done following a regular Capture-Kineticprotocol recommended by the manufacturer (Biacore GE Healthcare kineticwizard). Seven serial dilutions of human NKp46 recombinant proteins,ranging from 6.25 to 400 nM were sequentially injected over the capturedBi-Specific antibodies and allowed to dissociate for 10 min beforeregeneration. The entire sensorgram sets were fitted using the 1:1kinetic binding model.

Results

SPR showed that the bispecific polypeptides of format F1 having theNKp46-1, 2, 3 and 4 scFv binding domains bound to NKp46, while otherbispecific polypeptides having the scFv of other anti-NK46 antibodiesdid not retain NKp46 binding. The binding domains that did not retainbinding in monomeric bispecific format initially bound to NKp46 but lostbinding upon conversion to the bispecific format. All of the bispecificpolypeptides of formats F1, F2 F3, F4, F5, F6, F9, F10, F11, F13, F14retained binding to NKp46 when using the NKp46-3 variable regions.Monovalent affinities and kinetic association and dissociation rateconstants are shown below in the Table 3 below.

TABLE 3 Bispecific mAb ka (1/Ms) kd (1/s) KD (M) CD19-F1-Bab281 n/a n/an/a (loss of binding) CD19-F1-NKp46-1 1.23E+05 0.001337  1.09E−08CD19-F1-NKp46-2 1.62E+05 0.001445  8.93E−09 CD19-F1-NKp46-3 7.05E+046.44E−04  9.14E−09 CD19-F1-NKp46-4 1.35E+05 6.53E−04  4.85E−09CD19-F3-NKp46-3 3.905E+5 0.01117 28E−09 CD19-F4-NKp46-3 3.678E+5 0.0110030E−09 CD19-F5-NKp46-3 7.555E+4 0.00510 67E−09 CD19-F6-NKp46-3 7.934E+40.00503 63E−09 CD19-F9A-NKp46-3 2.070E+5 0.00669 32E−09CD19-F10A-NKp46-3 2.607E+5 0.00754 29E−09 CD19-F11A-NKp46-3 3.388E+50.01044 30E−09 CD19-F13-NKp46-3 8.300E+4 0.00565 68E−09 CD19-F14-NKp46-38.826E+4 0.00546 62E−09 NKp46-3 IgG1 2.224E+5 0.00433 20E−09

Example 6 Engagement of NK Cells Against Daudi Tumor Target withFc-Containing NKp46×CD19 Bispecific Protein

Bispecific antibodies having a monomeric Fc domain and a domainarrangement according to the single chain F1 or dimeric F2 formatsdescribed in Example 3, and a NKp46 binding region based on NKp46-1,NKp46-2, NKp46-3 or NKp46-4 were tested for functional ability to directNK cells to lyse CD19-positive tumor target cells (Daudi, a wellcharacterized B lymphoblast cell line). The F2 proteins additionallyincluded NKp46-9 variable regions which lost binding to NKp46 in thescFv format but which retained binding in the F(ab)-like format of F2.

Briefly, the cytolytic activity of each of (a) resting human NK cells,and (b) human NK cell line KHYG-1 transfected with human NKp46, wasassessed in a classical 4-h ⁵¹Cr-release assay in U-bottom 96 wellplates. Daudi cells were labelled with ⁵¹Cr (50 μCi (1.85 MBq)/1×10⁶cells), then mixed with KHYG-1 transfected with hNKp46 at aneffector/target ratio equal to 50 for KHYG-1, and 10 (for F1 proteins)or 8.8 (for F2 proteins) for resting NK cells, in the presence ofmonomeric bi-specific antibodies at different concentrations. Afterbrief centrifugation and 4 hours of incubation at 37° C., samples ofsupernatant were removed and transferred into a LumaPlate (Perkin ElmerLife Sciences, Boston, Mass.), and ⁵¹Cr release was measured with aTopCount NXT beta detector (PerkinElmer Life Sciences, Boston, Mass.).All experimental conditions were analyzed in triplicate, and thepercentage of specific lysis was determined as follows: 100×(mean cpmexperimental release−mean cpm spontaneous release)/(mean cpm totalrelease−mean cpm spontaneous release). Percentage of total release isobtained by lysis of target cells with 2% Triton X100 (Sigma) andspontaneous release corresponds to target cells in medium (withouteffectors or Abs).

Results

In the KHYG-1 hNKp46 NK experimental model, each bi-specific antibodyNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 induced specific lysis ofDaudi cells by human KHYG-1 hNKp46 NK cell line compared to negativecontrols (Human IgG1 isotype control (IC) and CD19/CD3 bi-specificantibodies), thereby showing that these antibodies induce Daudi targetcell lysis by KHYG-1 hNKp46 through CD19/NKp46 cross-linking.

When resting NK cells were used as effectors, each bi-specific antibodyNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 again induced specificlysis of Daudi cells by human NK cells compared to negative control(Human IgG1 isotype control (IC) antibody), thereby showing that theseantibodies induce Daudi target cell lysis by human NK cells throughCD19/NKp46 cross-linking. Rituximab (RTX, chimeric IgG1) was used as apositive control of ADCC (Antibody-Dependent Cell Cytotoxicity) byresting human NK cells. The maximal response obtained with RTX (at 10μg/ml in this assay) was 21.6% specific lysis illustrating that thebispecific antibodies have high target cell lysis activity. Results forexperiments with resting NK cells are shown in FIG. 3A for the singlechain F1 proteins and 3B for the dimeric F2 proteins.

Example 7 Comparison with Full Length Anti-NKp46 mAbs and DepletingAnti-Tumor mAbs: Only NKp46×CD19 Bispecific Proteins PreventNon-Specific NK Activation

These studies aimed to investigate whether bispecific antibodies canmediate NKp46-mediated NK activation toward cancer target cells withouttriggering non-specific NK cell activation.

NKp46×CD19 bispecific proteins having an arrangement according to the F2format described in Example 3 with anti-NKp46 variable domains fromNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 were compared to:

-   -   (a) full-length monospecific anti-NKp46 antibodies (NKp46-3 as        human IgG1), and    -   (b) the anti-CD19 antibody as a full-length human IgG1 as ADCC        inducing antibody control comparator.

The experiments further included as controls: rituximab, an anti-CD20ADCC inducing antibody control for a target antigen with high expressionlevels; anti-CD52 antibody alemtuzumab, a human IgG1, binds CD52 targetpresent on both targets and NK cells; and negative control isotypecontrol therapeutic antibody (a human IgG1 that does not bind a targetpresent on the target cells (HUG1-IC).

The different proteins were tested for functional effect on NK cellactivation in the presence of CD19-positive tumor target cells (Daudicells), in the presence of CD19-negative, CD16-positive target cells(HUT78 T-lymphoma cells), and in the absence of target cells.

Briefly, NK activation was tested by assessing CD69 and CD107 expressionon NK cells by flow cytometry. The assay was carried out in 96 U wellplates in completed RPM1, 150 μL final/well. Effector cells were freshNK cells purified from donors. Target cells were Daudi (CD19-positive),HUT78 (CD19-negative) or K562 (NK activation control cell line). Inaddition to K562 positive control, three conditions were tested, asfollows:

-   -   NK cell alone    -   NK cells vs Daudi (CD19+)    -   NK cells vs HUT78 (CD19−)

Effector:Target (E:T) ratio was 2.5:1 (50 000 E:20 000 T), with anantibody dilution range starting to 10 μg/mL with 1/4 dilution (n=8concentrations). Antibodies, target cells and effector cells were mixed;spun 1 min at 300 g; incubated 4 h at 37° C.; spun 3 min at 500 g;washed twice with Staining Buffer (SB); added 50 μL of staining Ab mix;incubated 30 min at 300 g; washed twice with SB resuspended pellet withCellFix; stored overnight at 4° C.; and fluorescence revealed with CantoII (HTS).

Results

1. NK Cells Alone

Results are shown in FIG. 4A. In the absence of target-antigenexpressing cells, none of the bispecific anti-NKp46×anti-CD19 antibody(including each of the NKp46-1, NKp46-2, NKp46-3, NKp46-4 and NKp46-9variable regions) activated NK cells as assessed by CD69 or CD107expression. Full-length anti-CD19 also did not activate NK cells.However, the full-length anti-NKp46 antibodies caused detectableactivation of NK cells. Alemtuzumab also induced activation of NK cells,at a very high level. Isotype control antibody did not induceactivation.

2. NK Cells vs Daudi (CD19+)

Results are shown in FIG. 4B. In the presence of target-antigenexpressing cells, each of the bispecific anti-NKp46×anti-CD19 antibodies(including each of the NKp46-1, NKp46-2, NKp46-3, NKp46-4 and NKp46-9binding domains) activated NK cells. Full-length anti-CD19 showed atbest only very low activation of NK cells. Neither full-lengthanti-NKp46 antibodies or alemtuzumab showed substantial increase inactivation beyond what was observed in presence of NK cells alone. FIG.4 shows full-length anti-NKp46 antibodies showed a similar level ofbaseline activation observed in presence of NK cells alone. Alemtuzumabalso induced activation of NK cells a similar level of activationobserved in presence of NK cells alone, and at higher antibodyconcentrations in this setting (ET 2.5:1) the activation was greaterthan with the bispecific anti-NKp46×anti-CD19 antibody. Isotype controlantibody did not induce activation.

3. NK Cells vs HUT78 (CD19−)

Results are shown in FIG. 4C. In the presence of target-antigen-negativeHUT78 cells, none of the bispecific anti-NKp46×anti-CD19 antibody(including each of the NKp46-1, NKp46-2, NKp46-3, NKp46-4 and NKp46-9variable regions) activated NK cells. However, the full-lengthanti-NKp46 antibodies and alemtuzumab caused detectable activation of NKcells at a similar level observed in presence of NK cells alone. Isotypecontrol antibody did not induce activation.

In conclusion, the bispecific anti-NKp46 proteins are able to activateNK cells in a target-cell specific manner, unlike full-lengthmonospecific anti-NKp46 antibodies and full-length antibodies ofdepleting IgG isotypes which also activate NK cells in the absence oftarget cells. The NK cell activation achieved with anti-NKp46 bispecificproteins was higher than that observed with full length anti-CD19 IgG1antibodies.

Example 8 Comparative Efficacy with Depleting Anti-Tumor mAbs:NKp46×CD19 Bispecific Proteins at Low ET Ratio

These studies aimed to investigate whether bispecific antibodies canmediate NKp46-mediated NK cell activation toward cancer target cells atlower effector:target ratios. The ET ratio used in this Example was 1:1which is believed to be closer to the setting that would be encounteredin vivo than the 2.5:1 ET ratio used in Example 7 or the 10:1 ET ratioof Example 6.

NKp46×CD19 bispecific proteins having an arrangement according to the F2format described in Example 3 with anti-NKp46 variable domains fromNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 were compared to:

-   -   (a) full-length monospecific anti-NKp46 antibodies (NKp46-3 as        human IgG1), and    -   (b) the anti-CD19 antibody as a full-length human IgG1 as ADCC        inducing antibody control comparator.

The experiments further included as controls: rituximab (an anti-CD20ADCC inducing antibody control for a target antigen with high expressionlevels); anti-CD52 antibody alemtuzumab (a human IgG1, binds CD52 targetpresent on both targets and NK cells), and negative control isotypecontrol therapeutic antibody (a human IgG1 that does not bind a targetpresent on the target cells (HUG1-IC). The different proteins weretested for functional effect on NK cell activation as assessed by CD69or CD107 expression in the presence of CD19-positive tumor target cells(Daudi cells), in the presence of CD19-negative, CD16-positive targetcells (HUT78 T-lymphoma cells), and in the absence of target cells. Theexperiments were carried out as in Example 7 except that the ET ratiowas 1:1.

Results

Results are shown in FIG. 5 (5A: CD107 and 5B: CD69). In the presence oftarget-antigen expressing cells, each of the bispecificanti-NKp46×anti-CD19 antibody (including each of the NKp46-1, NKp46-2,NKp46-3, NKp46-4 and NKp46-9 variable regions) activated NK cells in thepresence of Daudi cells.

The activation induced by bispecific anti-NKp46×anti-CD19 antibody inthe presence of Daudi cells was far more potent than the full-lengthhuman IgG1 anti-CD19 antibody as ADCC inducing antibody which had lowactivity in this setting. Furthermore, in this low E:T ratio setting theactivation induced by bispecific anti-NKp46×anti-CD19 antibody was aspotent as anti-CD20 antibody rituximab, with a difference being observedonly at the highest concentrations that were 10 fold higher thanconcentrations in which differences were observed at the 2.5:1 ET ratio.

In the absence of target cells or in the in the presence of targetantigen-negative HUT78 cells, full-length anti-NKp46 antibodies andalemtuzumab showed a similar level of baseline activation observed inthe presence of Daudi cells. Anti-NKp46×anti-CD19 antibody did notactivate NK cells in presence of HUT78 cells.

In conclusion, the bispecific anti-NKp46 proteins are able to activateNK cells in a target-cell specific manner and at lower effector:targetratio are more effective in mediating NK cell activation thattraditional human IgG1 antibodies.

Example 9 NKp46 Mechanism of Action

NKp46×CD19 bispecific proteins having an arrangement according to theF2, F3, F5 or F6 formats described in Examples 3 or 4 with anti-NKp46variable domains from NKp46-3 were compared to rituximab (anti-CD20 ADCCinducing antibody), and a human IgG1 isotype control antibody forfunctional ability to direct CD16-/NKp46+NK cell lines to lyseCD19-positive tumor target cells.

Briefly, the cytolytic activity of the CD16-/NKp46+ human NK cell lineKHYG-1 was assessed in a classical 4-h ⁵¹Cr-release assay in U-bottom 96well plates. Daudi or B221 cells were labelled with ⁵¹Cr (50 μCi (1.85MBq)/1×10⁶ cells), then mixed with KHYG-1 at an effector/target ratioequal to 50:1, in the presence of test antibodies at dilution rangestarting from 10⁻⁷ mol/L with 1/5 dilution (n=8 concentrations).

After brief centrifugation and 4 hours of incubation at 37° C., 50 μL ofsupernatant were removed and transferred into a LumaPlate (Perkin ElmerLife Sciences, Boston, Mass.), and ⁵¹Cr release was measured with aTopCount NXT beta detector (PerkinElmer Life Sciences, Boston, Mass.).All experimental conditions were analyzed in triplicate, and thepercentage of specific lysis was determined as follows: 100×(mean cpmexperimental release−mean cpm spontaneous release)/(mean cpm totalrelease−mean cpm spontaneous release). Percentage of total release isobtained by lysis of target cells with 2% Triton X100 (Sigma) andspontaneous release corresponds to target cells in medium (withouteffectors or Abs).

Results

Results are shown in FIG. 6A (KHYG-1 vs Daudi) and FIG. 6B (KHYG-1 vsB221). In the KHYG-1 hNKp46 NK experimental model, each NKp46×CD19bispecific protein (Format F2, F3, F5 and F6) induced specific lysis ofDaudi or B221 cells by human KHYG-1 hNKp46 NK cell line, while rituximaband human IgG1 isotype control (IC) antibodies did not.

Example 10 Comparative Efficacy of Bispecific and Conventional IgGAntibody Using Fresh Human NK Cells

NKp46×CD19 bispecific proteins that bind human CD16 having anarrangement according to the F5 format with anti-NKp46 variable domainsfrom NKp46-3 were compared to the same bispecific antibody as a F6format (which lacks CD16 binding), and to a human IgG1 isotype anti-CD19antibody, as well as a human IgG1 isotype control antibody forfunctional ability to direct purified NK cells to lyse CD19-positiveDaudi tumor target cells.

Briefly, the cytolytic activity of fresh human purified NK cells fromEFS Buffy Coat was assessed in a classical 4-h ⁵¹Cr-release assay inU-bottom 96 well plates. Daudi or HUT78 cells (negative control cellsthat do not express CD19) were labelled with ⁵¹Cr and then mixed with NKcells at an effector/target ratio equal to 10:1, in the presence of testantibodies at dilution range starting from 10 μg/ml with 1/10 dilution(n=8 concentrations).

After brief centrifugation and 4 hours of incubation at 37° C., 50 μL ofsupernatant were removed and transferred into a LumaPlate (Perkin ElmerLife Sciences, Boston, Mass.), and ⁵¹Cr release was measured with aTopCount NXT beta detector (PerkinElmer Life Sciences, Boston, Mass.).All experimental conditions were analyzed in triplicate, and thepercentage of specific lysis was determined as follows: 100×(mean cpmexperimental release−mean cpm spontaneous release)/(mean cpm totalrelease−mean cpm spontaneous release). Percentage of total release isobtained by lysis of target cells with 2% Triton X100 (Sigma) andspontaneous release corresponds to target cells in medium (withouteffectors or Abs).

Results are shown in FIG. 7 . The CD19-F6-NKp46 (bispecific protein inF6 format) whose Fc domain does not bind CD16 due to a N297 substitutionwas as potent in mediating NK cell lysis of Daudi target cells as thefull-length IgG1 anti-CD19 antibody, which is remarkable consideringthat the control IgG1 anti-CD19 antibody binds CD19 bivalently and thatthe anti-CD19 is bound by CD16. Surprisingly, the CD19-F6-NKp46 (F5format protein) whose Fc domain additionally binds CD16 is far morepotent in mediating Daudi target cell lysis that the full-length IgG1anti-CD19 antibody or the F6 format bispecific protein. At comparablelevels of target cell lysis, the CD19-F6-NKp46 was at least 1000 timesmore potent than the full-length anti-CD19 IgG1.

Example 11 Binding of Different Bispecific Formats to FcRn

Affinity of different antibody formats for human FcRn was studied bySurface Plasmon Resonance (SPR) by immobilizing recombinant FcRnproteins covalently to carboxyl groups in the dextran layer on a SensorChip CM5, as described in Example 2-6.

A chimeric full length anti-CD19 antibody having human IgG1 constantregions and NKp46×CD19 bispecific proteins having an arrangementaccording to the F3, F4, F5, F6, F9, F10, F11, F13 or F14 formatsdescribed in Examples 3 or 4 with anti-NKp46 variable domains fromNKp46-3 (NKp46-2 for F2) were tested; for each analyte, the entiresensorgram was fitted using the steady state or 1:1 SCK binding model.

Results are shown in Table 4 below. The bispecific proteins havingdimeric Fc domains (formats F5, F6, F13, F14) bound to FcRn withaffinity similar to that of the full-length IgG1 antibody. Thebispecific proteins with monomeric Fc domains (F3, F4, F9, F10, F11)also displayed binding to FcRn, however with lower affinity that thebispecific proteins having dimeric Fc domains.

TABLE 4 Antibody/Bispecific SPR method KD nM Human IgG1/K Anti-CD19SCK/Two state reaction 7.8 CD19-F5-NKp46-3 SCK/Two state reaction 2.6CD19-F6-NKp46-3 SCK/Two state reaction 6.0 CD19-F13-NKp46-3 SCK/Twostate reaction 15.2 CD19-F14-NKp46-3 SCK/Two state reaction 14.0CD19-F3-NKp46-3 Steady State 474.4 CD19-F4-NKp46-3 Steady State 711.7CD19-F9A-NKp46-3 Steady State 858.5 CD19-F10A-NKp46-3 Steady State 432.8CD19-F11-NKp46-3 Steady State 595.5

Example 12 Binding to FcγR

Different multimeric Fc proteins were evaluated to assess whether suchbispecific monomeric Fc protein could retain binding to Fcγ receptors.

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments HBS-EP+(Biacore GEHealthcare) and 10 mM NaOH, 500 mM NaCl served as running buffer andregeneration buffer respectively. Sensorgrams were analyzed with BiacoreT100 Evaluation software. Recombinant human FcRs (CD64, CD32a, CD32b,CD16a and CD16b) were cloned, produced and purified.

F5 and F6 bispecific antibodies CD19-F5-NKp46-3 or CD19-F6-NKp46-3 wereimmobilized covalently to carboxyl groups in the dextran layer on aSensor Chip CM5. The chip surface was activated with EDC/NHS(N-ethyl-N′-(3-dimethylaminopropyl) carbodiimidehydrochloride andN-hydroxysuccinimide (Biacore GE Healthcare)). Bispecific antibodieswere diluted to 10 μg/ml in coupling buffer (10 mM acetate, pH 5.6) andinjected until the appropriate immobilization level was reached (i.e.800 to 900 RU). Deactivation of the remaining activated groups wasperformed using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

Monovalent affinity study was assessed following a classical kineticwizard (as recommended by the manufacturer). Serial dilutions of solubleanalytes (FcRs) ranging from 0.7 to 60 nM for CD64 and from 60 to 5000nM for all the other FcRs were injected over the immobilized bispecificantibodies and allowed to dissociate for 10 min before regeneration. Theentire sensorgram sets were fitted using the 1:1 kinetic binding modelfor CD64 and with the Steady State Affinity model for all the otherFcRs.

While full length wild type human IgG1 bound to all cynomolgus and humanFcγ receptors, the CD19-F6-NKp46-3 bi-specific antibodies did not bindto any of the receptors. The CD19-F5-NKp46-3, on the other hand, boundto each of the human receptors CD64 (KD=0.7 nM), CD32a (KD=846 nM),CD32b (KD=1850 nM), CD16a (KD=1098 nM) and CD16b (KD=2426 nM).Convention human anti-IgG1 antibodies has comparable binding to the Fcreceptors (KD shown in the table below).

Full length human CD19-F5-NKp46-3 IgG1 antibody Human Fcγ receptor KD(nM) KD (nM) CD64 0.7 0.24 CD32a 846 379 CD32b 1850 1180 CD16a 1098 630CD16b 2426 2410

Example 13 Epitope Mapping of Anti-NKp46 Antibodies

A. Competition Assays

Competition assays were conducted by Surface Plasmon Resonance (SPRaccording to the methods described below.

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments HBS-EP+(Biacore GEHealthcare) and NaOH 10 mM NaCl 500 mM served as running buffer andregeneration buffer respectively. Sensorgrams were analyzed with BiacoreT100 Evaluation software. Anti-6×His tag antibody was purchased fromQIAGEN. Human 6×His tagged NKp46 recombinant proteins (NKp46-His) werecloned, produced and purified at Innate Pharma.

Anti-His antibodies were immobilized covalently to carboxyl groups inthe dextran layer on a Sensor Chip CM5. The chip surface was activatedwith EDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GEHealthcare)). Protein-A and Anti-His antibodies were diluted to 10 μg/mlin coupling buffer (10 mM acetate, pH 5.6) and injected until theappropriate immobilization level was reached (i.e. 2000 to 2500 RU).Deactivation of the remaining activated groups was performed using 100mM ethanolamine pH 8 (Biacore GE Healthcare).

Parental regular human IgG1 chimeric antibodies having NKp46 bindingregion corresponding to NKp46-1, NKp46-2, NKp46-3 or NKp46-4 were usedfor the competition study which has been performed using an Anti-6×Histag antibody chip.

Bispecific antibodies having NKp46 binding region based on NKp46-1,NKp46-2, NKp46-3 or NKp46-4 at 1 μg/mL were captured onto Protein-A chipand recombinant human NKp46 proteins were injected at 5 μg/mL togetherwith a second test bispecific antibody of the NKp46-1, NKp46-2, NKp46-3or NKp46-4 group.

None of NKp46-1, NKp46-2, NKp46-3 or NKp46-4 competed with one anotherfor binding to NKp46, these antibodies each representing a differentepitope.

B. Binding to NKp46 Mutants

In order to define the epitopes of anti NKp46 antibodies, we designedNKp46 mutants defined by one, two or three substitutions of amino acidsexposed at the molecular surface over the 2 domains of NKp46. Thisapproach led to the generation of 42 mutants transfected in Hek-293Tcells, as shown in the table below. The targeted amino acid mutations inthe table 5 below are shown both using numbering of SEQ ID NO: 1 (alsocorresponding to the numbering used in Jaron-Mendelson et al. (2012) J.Immunol. 88(12):6165-74.

TABLE 5 Substitution (Numbering according to: Mutant Jaron-Mendelson andSEQ ID NO 1) 1 P40A K43S Q44A 2 K41S E42A E119A 3 P86A D87A 4 N89A R91A5 K80A K82A  5bis E34A T46A 6 R101A V102A 7 N52A Y53A 8 V56A P75A E76A 9R77A I78A 10 S97A I99A 10bis Q59A H61A 11 L66A V69A 12 E108A 13 N111AL112A 14 D114A 15 T125A R145S D147A 16 S127A Y143A 17 H129A K139A 18K170A V172A 19 I135A S136A 19bis T182A R185A 20 R160A 21 K207A 22 M152AR166A 23 N195A N196A Stalk1 D213A I214A T217A Stalk2 F226A T233A Stalk3L236A T240A Supp1 F30A W32A Supp2 F62A F67A Supp3 E63A Q95A Supp4 R71AK73A Supp5 Y84A Supp6 E104A L105A Supp7 Y121A Y194A Supp8 P132A E133ASupp9 S151A Y168A  Supp10 S162A H163A  Supp11 E174A P176A  Supp12 P179AH184A  Supp13 R189A E204A P205AGeneration of Mutants

NKp46 mutants were generated by PCR. The sequences amplified were run onagarose gel and purified using the Macherey Nagel PCR Clean-Up GelExtraction kit (reference 740609). The two or three purified PCRproducts generated for each mutant were then ligated into an expressionvector, with the ClonTech InFusion system. The vectors containing themutated sequences were prepared as Miniprep and sequenced. Aftersequencing, the vectors containing the mutated sequences were preparedas Midiprep using the Promega PureYield™ Plasmid Midiprep System.HEK293T cells were grown in DMEM medium (Invitrogen), transfected withvectors using Invitrogen's Lipofectamine 2000 and incubated at 37° C. ina CO2 incubator for 24 hours prior to testing for transgene expression.

Flow Cytometry Analysis of Anti-NKp46 Binding to the HEK293T TransfectedCells

All the anti-NKp46 antibodies were tested for their binding to eachmutant by flow cytometry. A first experiment was performed to determineantibodies that lose their binding to one or several mutants at oneconcentration (10 μg/ml). To confirm a loss of binding, titration ofantibodies was done on antibodies for which binding seemed to beaffected by the NKp46 mutations (1-0.1-0.01-0.001 μg/ml).

Results

Antibody NKp46-1 had decreased binding to the mutant 2 (having amutation at residues K41, E42 and E119 (numbering in NKp46 wild-type)compared to wild-type NK46. Similarly, NKp46-1 also had decreasedbinding to the supplementary mutant Supp7 (having a mutation at residuesY121 and Y194.

Antibody NKp46-3 had decreased binding to the mutant 19 (having amutation at residues 1135, and S136. Similarly, NKp46-1 also haddecreased binding to the supplementary mutant Supp8 (having a mutationat residues P132 and E133.

Antibody NKp46-4 had decreased binding to the mutant 6 (having amutation at residues R101, and V102. Similarly, NKp46-1 also haddecreased binding to the supplementary mutant Supp6 having a mutation atresidues E104 and L105.

In this study, we identified epitopes for anti-NKp46 antibodies(NKp46-1, NKp46-3 and NKp46-4). Epitopes of NKp46-4, NKp46-3 and NKp46-1are on NKp46 D1 domain, D2 domain and D1/D2 junction, respectively.R101, V102, E104 and L105 are essential residues for NKp46-4 binding anddefined a part of NKp46-4 epitope. The epitope of NKp46-1 epitopeincludes K41, E42, E119, Y121 and Y194 residues. The epitope of NKp46-3includes P132, E133, 1135, and S136 residues.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way. Anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e. g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Allmethods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as reference to an element or elements is intended toprovide support for a similar aspect or embodiment of the invention that“consists of,” “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context (e.g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

All publications and patent applications cited in this specification areherein incorporated by reference in their entireties as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

We claim:
 1. An isolated nucleic acid encoding a heavy chain variableregion (VH) and/or a light chain variable region (VL) selected from thegroup consisting of: (a) a VH comprising an amino acid sequence of SEQID NOS: 199 or 200 (NKp46-1 H1 or H3 variable domain), and a VLcomprising an amino acid sequence of the amino acid sequence of SEQ IDNO: 201 (NKp46-1 L1 variable domain); (b) a VH comprising an amino acidsequence of SEQ ID NOS: 202, 203 or 204 (NKp46-2 H1, H2 or H3 variabledomain), and a VL comprising an amino acid sequence of SEQ ID NO: 205(NKp46-2 L1 variable domain); (c) a VH comprising an amino acid sequenceof SEQ ID NOS: 206, 207 or 208 (NKp46-3 H1, H3 or H4 variable domain),and a VL comprising an amino acid sequence of SEQ ID NO: 209 (NKp46-3 L1variable domain); (d) a VH comprising an amino acid sequence of SEQ IDNOS: 210, 211 or 212 (NKp46-4 H1 variable domain), and a VL comprisingan amino acid sequence of SEQ ID NO: 213 (NKp46-4 L2 variable domain);(e) a VH comprising an amino acid sequence of SEQ ID NO: 215 (NKp46-9 H2variable domain), and a VL comprising an amino acid sequence of SEQ IDNOS: 217 or 218 (NKp46-9 L1 or L2 variable domain); or (f) a VHcomprising an amino acid sequence of SEQ ID NO: 216 (NKp46-9 H3 variabledomain), and a VL comprising an amino acid sequence of SEQ ID NOS: 217or 218 (NKp46-9 L1 or L2 variable domain).
 2. A recombinant host cellcomprising a nucleic acid according to claim
 1. 3. A method of producinga protein comprising culturing a cell of claim 2 under conditionssuitable for expression of the protein and recovering the protein. 4.The nucleic acid according to claim 1, wherein said nucleic acid encodesa VH comprising SEQ ID NOS: 199 or
 200. 5. The nucleic acid according toclaim 1, wherein said nucleic acid encodes a VL comprising SEQ ID NO:201.
 6. The nucleic acid according to claim 1, wherein said nucleic acidencodes a VH comprising SEQ ID NOS: 202, 203 or
 204. 7. The nucleic acidaccording to claim 1, wherein said nucleic acid encodes a VL comprisingSEQ ID NO:
 205. 8. The nucleic acid according to claim 1, wherein saidnucleic acid encodes a VH comprising SEQ ID NOS: 206, 207 or
 208. 9. Thenucleic acid according to claim 1, wherein said nucleic acid encodes aVL comprising SEQ ID NO:
 209. 10. The nucleic acid according to claim 1,wherein said nucleic acid encodes a VH comprising SEQ ID NOS: 210, 211or
 212. 11. The nucleic acid according to claim 1, wherein said nucleicacid encodes a VL comprising SEQ ID NO:
 213. 12. The nucleic acidaccording to claim 1, wherein said nucleic acid encodes a VH comprisingSEQ ID NO:
 215. 13. The nucleic acid according to claim 1, wherein saidnucleic acid encodes a VL comprising SEQ ID NOS: 217 or
 218. 14. Thenucleic acid according to claim 1, wherein said nucleic acid encodes aVH comprising SEQ ID NO:
 216. 15. The nucleic acid according to claim 1,wherein said nucleic acid encodes a VL comprising SEQ ID NOS: 217 or218.